Lateral flow assays using dna dendrimers

ABSTRACT

The present invention relates to lateral flow devices and kits using DNA dendrimers, and to methods for detecting an analyte using the lateral flow devices and kits. More specifically, the DNA dendrimers may be dried on a location on the test device upstream from said test zone, before said test device is used.

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. provisionalapplication Ser. No. 61/671,662, filed Jul. 13, 2012, entitled “LATERALFLOW ASSAYS USING DNA DENDRIMERS” and U.S. utility application Ser. No.13/844,601, filed Mar. 15, 2013, entitled “LATERAL FLOW ASSAYS USING DNADENDRIMERS.” The contents of the above-referenced applications areincorporated by reference in their entireties.

TECHNICAL FIELD

The present invention relates to novel lateral flow devices and kitsusing DNA dendrimers, and the methods for detecting an analyte using thelateral flow devices and kits.

BACKGROUND OF THE INVENTION

The 3DNA® DNA dendrimer is a proprietary dendritic molecule comprisedsolely of DNA. As a class, dendrimers are complex, highly branchedmolecules built from interconnected natural or synthetic monomericsubunits. A DNA dendrimer is constructed from DNA monomers, each ofwhich is made from two DNA strands that share a region of sequencecomplementarity located in the central portion of each strand (FIG. 1).Monomers are combined during the manufacturing process to prepare DNAdendrimers of different sizes and shapes (FIG. 2). In order to preventDNA dendrimers from “falling apart” over time, chemical “spot welds” aresometimes added to the growing assembly during the process using UVlight via the intercalation and activation of psoralen cross-linkers.Dendrimers have been historically purified according to their size andmolecular weight on denaturing sucrose gradients afterultracentrifugation.

DNA dendrimers have previously been used in membrane based assays,specifically for the detection of nucleic acids and proteinsnon-covalently immobilized to various membrane substrates, includingnitrocellulose and nylon. These assays typically required the use ofdendrimers specifically derivatized to contain targeting or bindingmoieties specific for the target analyte, and typically required severalhours to overnight for optimal binding. Improvement of sensitivity fromsignal amplification ranged from 5 to 500 fold over the non-dendrimerversion of the assay.

DNA dendrimers have also been shown to be useful as signal amplifiers ina number of other applications, including nucleic acid (DNA/RNA)microarrays, ELISAs, ELOSAs, bead based immunoassays, protein arrays andother similar assays. These assays are all characterized by theimmobilization of the analyte or target material to a substrate eitherdirectly via a non-covalent or a covalent binding process, or indirectlyvia the binding to a previously immobilized ligand, which generallyrequired several steps prior to or during the assay process. DNAdendrimers containing up to hundreds of label moieties would then bindeither directly or indirectly to the analyte via a targeting devicewhich would simultaneously directly bind to both the analyte (orindirectly to a ligand bound to the analyte) and the dendrimer, therebycreating a bridge between the multi-labeled dendrimer and the analyte.Signal was generated in these assays either directly from label moietieson the dendrimer (e.g. fluorescent dyes) or indirectly via the bindingof signaling moieties to binding sites on the dendrimer (e.g.streptavidin-HRP binding to dendrimer bound biotins).

Therefore, there is a need for devices and methods that overcome thelimitations of the current technologies and methodologies, especiallyfor more sensitive lateral flow assays. The present invention addressesthese and other related needs.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides for a test device fordetecting an analyte in a liquid sample, which device comprises a porousmatrix that comprises a test zone on said porous matrix, said test zonecomprising a test reagent that binds to an analyte or another bindingreagent that binds to said analyte, or is an analyte or an analyteanalog that competes with an analyte in said sample for binding to abinding reagent for said analyte, wherein a liquid sample flowslaterally along said test device and passes (or a liquid sample iscapable of flowing laterally along said test device and passing) saidtest zone to form a detectable signal to indicate presence, absenceand/or amount of said analyte in said liquid sample, the formation ofsaid detectable signal requires the use of a detectable label and a DNAdendrimer, said DNA dendrimer comprises a first component that linkssaid DNA dendrimer to a binding reagent linkable to said DNA dendrimer,an analyte linkable to said DNA dendrimer, or an analyte analog linkableto said DNA dendrimer, and a second component that links said DNAdendrimer to said detectable label linkable to said DNA dendrimer.

In some embodiments, the analyte is not a polynucleotide; or the DNAdendrimer comprises from about 1 to about 324, preferably from about 60to about 324, from about 80 to about 300 or from about 100 to about 200,the first components; or the DNA dendrimer, before the test device isused, is dried on a location on the test device upstream from the testzone. In some embodiments, the test reagent or binding reagentspecifically binds to an analyte.

In another aspect, the present disclosure provides for a method fordetecting an analyte in a liquid sample, which method comprises: a)contacting a liquid sample with the above test device, wherein theliquid sample is applied to a site of the test device upstream of thetest zone; b) transporting an analyte, if present in the liquid sample,a detectable label and a DNA dendrimer to the test zone; and c)assessing the presence, absence, and/or amount of a signal generated bythe detectable label at the test zone to determine the presence, absenceand/or amount of the analyte in the liquid sample.

In still another aspect, the present disclosure provides for a testdevice for detecting an analyte in a liquid sample, which devicecomprises a porous matrix that comprises a test zone on said porousmatrix, said test zone comprising a test reagent that binds to ananalyte or another binding reagent that binds to said analyte, or is ananalyte or an analyte analog that competes with an analyte in saidsample for binding to a binding reagent for said analyte, wherein aliquid sample flows laterally along said test device and passes (or aliquid sample is capable of flowing laterally along said test device andpassing) said test zone to form a detectable signal to indicatepresence, absence and/or amount of said analyte in said liquid sample,the formation of said detectable signal requires the use of a DNAdendrimer linked to a detectable label covalently or non-covalently, andsaid DNA dendrimer comprises a component that links said DNA dendrimerto a binding reagent linkable to said DNA dendrimer, an analyte linkableto said DNA dendrimer, or an analyte analog linkable to said DNAdendrimer.

In some embodiments, the analyte is not a polynucleotide; or the DNAdendrimer comprises from about 1 to about 324, preferably from about 60to about 324, from about 80 to about 300 or from about 100 to about 200,the first components; or the DNA dendrimer, before the test device isused, is dried on a location on the test device upstream from the testzone. In some embodiments, the test reagent or binding reagentspecifically binds to an analyte.

In yet another aspect, the present disclosure provides for a method fordetecting an analyte in a liquid sample, which method comprises: a)contacting a liquid sample with the above test device, wherein theliquid sample is applied to a site of the test device upstream of thetest zone; b) transporting an analyte, if present in the liquid sample,a detectable label and a DNA dendrimer to the test zone; and c)assessing the presence, absence, and/or amount of a signal generated bythe detectable label at the test zone to determine the presence, absenceand/or amount of the analyte in the liquid sample.

In yet another aspect, the present disclosure provides for a test devicefor detecting an analyte in a liquid sample, which device comprises aporous matrix that comprises a test zone on said porous matrix, saidtest zone comprising a test reagent that binds to an analyte or anotherbinding reagent that binds to said analyte, or is an analyte or ananalyte analog that competes with an analyte in said sample for bindingto a binding reagent for said analyte, wherein a liquid sample iscapable of flowing laterally along said test device and passing saidtest zone to form a detectable signal to indicate presence, absenceand/or amount of said analyte in said liquid sample, wherein: a) theformation of said detectable signal requires the use of a detectablelabel and a DNA dendrimer, said DNA dendrimer comprises a firstcomponent that links said DNA dendrimer to a binding reagent linkable tosaid DNA dendrimer, an analyte linkable to said DNA dendrimer, or ananalyte analog linkable to said DNA dendrimer, and a second componentthat links said DNA dendrimer to said detectable label linkable to saidDNA dendrimer, or b) the formation of said detectable signal requiresthe use of a DNA dendrimer linked to a detectable label covalently ornon-covalently, and said DNA dendrimer comprises a third component thatlinks said DNA dendrimer to a binding reagent linkable to said DNAdendrimer, an analyte linkable to said DNA dendrimer, or an analyteanalog linkable to said DNA dendrimer, and wherein: c) said analyte isnot a polynucleotide; and/or d) said DNA dendrimer comprises from about1 to about 324, preferably from about 60 to about 324, from about 80 toabout 300 or from about 100 to about 200, said first components; and/ore) said DNA dendrimer, before said test device is used, is dried on alocation on said test device upstream from said test zone. Methods fordetecting an analyte in a liquid sample using the above device are alsoprovided.

In yet another aspect, the present disclosure provides for a kit fordetecting an analyte in a liquid sample, which kit comprises: a) aporous matrix that comprises a test zone on said porous matrix, saidtest zone comprising a test reagent that binds to an analyte or anotherbinding reagent that binds to said analyte, or is an analyte or ananalyte analog that competes with an analyte in said sample for bindingto a binding reagent for said analyte; and b) a DNA dendrimer,wherein: 1) said DNA dendrimer comprises a first component that linkssaid DNA dendrimer to a binding reagent linkable to said DNA dendrimer,an analyte linkable to said DNA dendrimer, or an analyte analog linkableto said DNA dendrimer, and a second component that links said DNAdendrimer to said detectable label linkable to said DNA dendrimer, or 2)said DNA dendrimer is linked to a detectable label covalently ornon-covalently and comprises a third component that links said DNAdendrimer to a binding reagent linkable to said DNA dendrimer, ananalyte linkable to said DNA dendrimer, or an analyte analog linkable tosaid DNA dendrimer.

The principles of the present test devices, kits and methods can beapplied, or can be adapted to apply, to the lateral flow test devicesand assays known in the art. For example, the principles of the presenttest devices and methods can be applied, or can be adapted to apply, tothe lateral flow test devices and assays disclosed and/or claimed in theU.S. Pat. Nos. 3,641,235, 3,959,078, 3,966,897, 4,094,647, 4,168,146,4,299,916, 4,347,312, 4,366,241, 4,391,904, 4,425,438, 4,517,288,4,960,691, 5,141,875, 4,857,453, 5,073,484, 4,695,554, 4,703,017,4,743,560, 5,075,078, 5,591,645, 5,656,448, RE 38,430 E, 5,602,040,6,017,767, 6,319,676, 6,352,862, 6,485,982, 5,120,643, 4,956,302, RE39,664 E, 5,252,496, 5,514,602, 7,238,538 B2, 7,175,992 B2, 6,770,487B2, 5,712,170, 5,275,785, 5,504,013, 6,156,271, 6,187,269, 6,399,398,7,317,532, EP 0,149,168 A1, EP 0,323,605 A1, EP 0,250,137 A2, GB1,526,708 and WO99/40438.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 illustrates an exemplary, standard lateral flow “point of care”assay format.

FIG. 2 illustrates an exemplary dendrimer enhanced lateral flow “pointof care” assay format. Dendrimer-antibody reagents are supplied drieddown on the conjugate pad. During the lateral flow assay, dendrimerscollect and concentrate high concentrations of gold particles at thetest line, thereby increasing the signal intensity for captured proteinmolecules over the standard non-dendrimer assay.

FIG. 3 illustrates an exemplary manufacturing DNA dendrimers: Stepwise“assembly” of the dendrimer structure. Dendrimers are assembled from 5partially double stranded “monomers” added in a controlled step-by-stepprocess. In some embodiments, the final dendrimer matrix structure isfully covalent via the addition of a psorlen crosslinking intercalationreagent during the assembly process.

FIG. 4 illustrates an exemplary dendrimer molecule containing targetingantibodies and labels for immunoassays.

FIG. 5( a) illustrates protein detection by 3DNA Dendrimers in a modelhCG lateral flow POC assay (direct sandwich method).

FIG. 5( b) illustrates DNA dendrimer, anti-hCG antibody-oligo conjugateand gold reagents were added as liquid components during the performanceof an exemplary dendrimer lateral flow assay. In some embodiments, 3DNADendrimer assay is 16 fold more sensitive than anti-hCG gold standardassay, and ˜32 fold more sensitive than biotinylated anti-hCG SA-goldstandard assay.

FIG. 5( c) illustrates comparison of 3DNA Dendrimer “wet” assay to twostandard assays.

FIG. 6( a) illustrates protein detection by 3DNA Dendrimers in a modelhCG lateral flow POC assay (indirect sandwich method).

FIG. 6( b) illustrates DNA dendrimer, anti-hCG antibody-oligo conjugate,native antibody and gold reagents were added as liquid components duringthe performance of an exemplary dendrimer lateral flow assay. In someembodiments, 3DNA Dendrimer Assay is 16 fold more sensitive thananti-hCG Gold Standard assay, and ˜32 fold more sensitive thanbiotinylated anti-hCG SA-Gold Standard assay.

FIG. 7 illustrates conversion of the multistep Dendrimer LF assay to anexemplary 1-step fully dried down assay.

FIG. 8( a) illustrates lateral flow protein detection by exemplaryfluorescent 3DNA Dendrimers: Comparison to directly labeled fluorescentantibody.

FIG. 8( b) illustrates lateral flow (LF) protein detection by exemplaryfluorescent 3DNA Dendrimers: Comparison to directly labeled fluorescentantibody.

FIG. 9 illustrates the lateral flow devices used in Example 10.

FIG. 10 illustrates the results of dendrimer dried withoutantibody-oligo conjugate and run on HF90 nitrocellulose containing hCGlateral flow assays.

FIG. 11 illustrates the results of dendrimer dried withoutantibody-oligo conjugate and run on HF180 nitrocellulose containing hCGlateral flow assays.

FIG. 12 illustrates the results of dendrimer dried with antibody-oligoconjugate and performed on HF90 nitrocellulose containing hCG lateralflow assays.

FIG. 13 illustrates the DCN scoring system.

FIG. 14 illustrates the test results in Example 11.

DETAILED DESCRIPTION OF THE INVENTION

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections thatfollow.

A. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications referred to herein areincorporated by reference in their entireties. If a definition set forthin this section is contrary to or otherwise inconsistent with adefinition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth in this section prevails over thedefinition that is incorporated herein by reference.

As used herein, “a” or “an” means “at least one” or “one or more.”

As used herein, a “binding reagent” refers to any substance that bindsto target or analyte with desired affinity and/or specificity.Non-limiting examples of the binding reagent include cells, cellularorganelles, viruses, particles, microparticles, molecules, or anaggregate or complex thereof, or an aggregate or complex of molecules.Exemplary binding reagents can be an amino acid, a peptide, a protein,e.g., an antibody or receptor, a nucleoside, a nucleotide, anoligonucleotide, a nucleic acid, e.g., DNA or RNA, a vitamin, amonosaccharide, an oligosaccharide, a carbohydrate, a lipid, an aptamerand a complex thereof.

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule, and can be animmunoglobulin of any class, e.g., IgG, IgM, IgA, IgD and IgE. IgY,which is the major antibody type in avian species such as chicken, isalso included within the definition. As used herein, the termencompasses not only intact polyclonal or monoclonal antibodies, butalso fragments thereof (such as Fab, Fab′, F(ab′)2, Fv), single chain(ScFv), mutants thereof, naturally occurring variants, fusion proteinscomprising an antibody portion with an antigen recognition site of therequired specificity, humanized antibodies, chimeric antibodies, and anyother modified configuration of the immunoglobulin molecule thatcomprises an antigen recognition site of the required specificity.

As used herein, “monoclonal antibody” refers to an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theantibodies comprising the population are identical except for possiblenaturally occurring mutations that are present in minor amounts. As usedherein, a “monoclonal antibody” further refers to functional fragmentsof monoclonal antibodies.

As used herein, the term “antigen” refers to a target molecule that isspecifically bound by an antibody through its antigen recognition site.The antigen may be monovalent or polyvalent, i.e., it may have one ormore epitopes recognized by one or more antibodies. Examples of kinds ofantigens that can be recognized by antibodies include polypeptides,oligosaccharides, glycoproteins, polynucleotides, lipids, etc.

As used herein, the term “specifically binds” refers to the specificityof a binding reagent, e.g., an antibody, such that it preferentiallybinds to a defined analyte or target. Recognition by a binding reagentor an antibody of a particular analyte or target in the presence ofother potential interfering substance(s) is one characteristic of suchbinding. In some embodiments, a binding reagent that specifically bindsto an analyte avoids binding to other interfering moiety or moieties inthe sample to be tested.

As used herein the term “avoids binding” refers to the specificity ofparticular binding reagents, e.g., antibodies or antibody fragments.Binding reagents, antibodies or antibody fragments that avoid binding toa particular moiety generally contain a specificity such that a largepercentage of the particular moiety would not be bound by such bindingreagents, antibodies or antibody fragments. This percentage generallylies within the acceptable cross reactivity percentage with interferingmoieties of assays utilizing the binding reagents or antibodies directedto detecting a specific target. Frequently, the binding reagents,antibodies or antibody fragments of the present disclosure avoid bindinggreater than about 90% of an interfering moiety, although higherpercentages are clearly contemplated and preferred. For example, bindingreagents, antibodies or antibody fragments of the present disclosureavoid binding about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, about 99%, and about 99% or more of aninterfering moiety. Less occasionally, binding reagents, antibodies orantibody fragments of the present disclosure avoid binding greater thanabout 70%, or greater than about 75%, or greater than about 80%, orgreater than about 85% of an interfering moiety.

As used herein, “mammal” refers to any of the mammalian class ofspecies. Frequently, the term “mammal,” as used herein, refers tohumans, human subjects or human patients.

As used herein, the term “subject” is not limited to a specific speciesor sample type. For example, the term “subject” may refer to a patient,and frequently a human patient. However, this term is not limited tohumans and thus encompasses a variety of mammalian species.

As used herein the term “sample” refers to anything which may contain ananalyte for which an analyte assay is desired. The sample may be abiological sample, such as a biological fluid or a biological tissue.Examples of biological fluids include urine, blood, plasma, serum,saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus,amniotic fluid or the like. Biological tissues are aggregate of cells,usually of a particular kind together with their intercellular substancethat form one of the structural materials of a human, animal, plant,bacterial, fungal or viral structure, including connective, epithelium,muscle and nerve tissues. Examples of biological tissues also includeorgans, tumors, lymph nodes, arteries and individual cell(s).

As used herein the term “isolated” refers to material removed from itsoriginal environment, and is altered from its natural state. Forexample, an isolated polypeptide could be coupled to a carrier, andstill be “isolated” because that polypeptide is not in its originalenvironment.

As used herein, high-throughput screening (HTS) refers to processes thattest a large number of samples, such as samples of diverse chemicalstructures against disease targets to identify “hits” (see, e.g.,Broach, et al., High throughput screening for drug discovery, Nature,384:14-16 (1996); Janzen, et al., High throughput screening as adiscovery tool in the pharmaceutical industry, Lab Robotics Automation:8261-265 (1996); Fernandes, P. B., Letter from the society president, J.Biomol. Screening, 2:1 (1997); Burbaum, et al., New technologies forhigh-throughput screening, Curr. Opin. Chem. Biol., 1:72-78 (1997)). HTSoperations are highly automated and computerized to handle samplepreparation, assay procedures and the subsequent processing of largevolumes of data.

The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” areused interchangeably herein to refer to polymers of amino acids of anylength, e.g., at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300,400, 500, 1,000 or more amino acids. The polymer may be linear orbranched, it may comprise modified amino acids, and it may beinterrupted by non-amino acids. The terms also encompass an amino acidpolymer that has been modified naturally or by intervention; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation or modification,such as conjugation with a labeling component. Also included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),as well as other modifications known in the art.

The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and“nucleic acid molecule” are used interchangeably herein to refer to apolymeric form of nucleotides of any length, e.g., at least 8, 9, 10,20, 30, 40, 50, 100, 200, 300, 400, 500, 1,000 or more nucleotides, andmay comprise ribonucleotides, deoxyribonucleotides, analogs thereof, ormixtures thereof. This term refers only to the primary structure of themolecule. Thus, the term includes triple-, double- and single-strandeddeoxyribonucleic acid (“DNA”), as well as triple-, double- andsingle-stranded ribonucleic acid (“RNA”). It also includes modified, forexample by alkylation, and/or by capping, and unmodified forms of thepolynucleotide. More particularly, the terms “polynucleotide,”“oligonucleotide,” “nucleic acid” and “nucleic acid molecule” includepolydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA,and mRNA, whether spliced or unspliced, any other type of polynucleotidewhich is an N- or C-glycoside of a purine or pyrimidine base, and otherpolymers containing normucleotidic backbones, for example, polyamide(e.g., peptide nucleic acids (“PNAs”)) and polymorpholino (commerciallyavailable from the Anti-Virals, Inc., Corvallis, Oreg., as Neugene)polymers, and other synthetic sequence-specific nucleic acid polymersproviding that the polymers contain nucleobases in a configuration whichallows for base pairing and base stacking, such as is found in DNA andRNA. Thus, these terms include, for example, 3′-deoxy-2′,5′-DNA,oligodeoxyribonucleotide N3′ to P5′ phosphoramidates,2′-O-alkyl-substituted RNA, hybrids between DNA and RNA or between PNAsand DNA or RNA, and also include known types of modifications, forexample, labels, alkylation, “caps,” substitution of one or more of thenucleotides with an analog, intemucleotide modifications such as, forexample, those with uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoramidates, carbamates, etc.), with negativelycharged linkages (e.g., phosphorothioates, phosphorodithioates, etc.),and with positively charged linkages (e.g., aminoalkylphosphoramidates,aminoalkylphosphotriesters), those containing pendant moieties, such as,for example, proteins (including enzymes (e.g. nucleases), toxins,antibodies, signal peptides, poly-L-lysine, etc.), those withintercalators (e.g., acridine, psoralen, etc.), those containingchelates (of, e.g., metals, radioactive metals, boron, oxidative metals,etc.), those containing alkylators, those with modified linkages (e.g.,alpha anomeric nucleic acids, etc.), as well as unmodified forms of thepolynucleotide or oligonucleotide.

It will be appreciated that, as used herein, the terms “nucleoside” and“nucleotide” will include those moieties which contain not only theknown purine and pyrimidine bases, but also other heterocyclic baseswhich have been modified. Such modifications include methylated purinesor pyrimidines, acylated purines or pyrimidines, or other heterocycles.Modified nucleosides or nucleotides can also include modifications onthe sugar moiety, e.g., wherein one or more of the hydroxyl groups arereplaced with halogen, aliphatic groups, or are functionalized asethers, amines, or the like. The term “nucleotidic unit” is intended toencompass nucleosides and nucleotides.

“Nucleic acid probe” and “probe” are used interchangeably and refer to astructure comprising a polynucleotide, as defined above, that contains anucleic acid sequence that can bind to a corresponding target. Thepolynucleotide regions of probes may be composed of DNA, and/or RNA,and/or synthetic nucleotide analogs.

As used herein, “complementary or matched” means that two nucleic acidsequences have at least 50% sequence identity. Preferably, the twonucleic acid sequences have at least 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% of sequence identity. “Complementary or matched” alsomeans that two nucleic acid sequences can hybridize under low, middleand/or high stringency condition(s).

As used herein, “substantially complementary or substantially matched”means that two nucleic acid sequences have at least 90% sequenceidentity. Preferably, the two nucleic acid sequences have at least 95%,96%, 97%, 98%, 99% or 100% of sequence identity. Alternatively,“substantially complementary or substantially matched” means that twonucleic acid sequences can hybridize under high stringency condition(s).

In general, the stability of a hybrid is a function of the ionconcentration and temperature. Typically, a hybridization reaction isperformed under conditions of lower stringency, followed by washes ofvarying, but higher, stringency. Moderately stringent hybridizationrefers to conditions that permit a nucleic acid molecule such as a probeto bind a complementary nucleic acid molecule. The hybridized nucleicacid molecules generally have at least 60% identity, including forexample at least any of 70%, 75%, 80%, 85%, 90%, or 95% identity.Moderately stringent conditions are conditions equivalent tohybridization in 50% formamide, 5×Denhardt's solution, 5×SSPE, 0.2% SDSat 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 42° C. Highstringency conditions can be provided, for example, by hybridization in50% formamide, 5×Denhardt's solution, 5×SSPE, 0.2% SDS at 42° C.,followed by washing in 0.1×SSPE, and 0.1% SDS at 65° C. Low stringencyhybridization refers to conditions equivalent to hybridization in 10%formamide, 5×Denhardt's solution, 6×SSPE, 0.2% SDS at 22° C., followedby washing in 1×SSPE, 0.2% SDS, at 37° C. Denhardt's solution contains1% Ficoll, 1% polyvinylpyrolidone, and 1% bovine serum albumin (BSA).20×SSPE (sodium chloride, sodium phosphate, ethylene diamide tetraaceticacid (EDTA)) contains 3M sodium chloride, 0.2M sodium phosphate, and0.025 M EDTA. Other suitable moderate stringency and high stringencyhybridization buffers and conditions are well known to those of skill inthe art and are described, for example, in Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press,Plainview, N.Y. (1989); and Ausubel et al., Short Protocols in MolecularBiology, 4th ed., John Wiley & Sons (1999).

Alternatively, substantial complementarity exists when an RNA or DNAstrand will hybridize under selective hybridization conditions to itscomplement. Typically, selective hybridization will occur when there isat least about 65% complementary over a stretch of at least 14 to 25nucleotides, preferably at least about 75%, more preferably at leastabout 90% complementary. See Kanehisa (1984) Nucleic Acids Res.12:203-215.

As used herein, “biological sample” refers to any sample obtained from aliving or viral source or other source of macromolecules andbiomolecules, and includes any cell type or tissue of a subject fromwhich nucleic acid or protein or other macromolecule can be obtained.The biological sample can be a sample obtained directly from abiological source or a sample that is processed. For example, isolatednucleic acids that are amplified constitute a biological sample.Biological samples include, but are not limited to, body fluids, such asblood, plasma, serum, cerebrospinal fluid, synovial fluid, urine andsweat, tissue and organ samples from animals and plants and processedsamples derived therefrom. Also included are soil and water samples andother environmental samples, viruses, bacteria, fungi, algae, protozoaand components thereof.

It is understood that aspects and embodiments of the invention describedherein include “consisting” and/or “consisting essentially of” aspectsand embodiments.

Throughout this disclosure, various aspects of this invention arepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible sub-ranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Other objects, advantages and features of the present invention willbecome apparent from the following specification taken in conjunctionwith the accompanying drawings.

B. LATERAL ASSAY DEVICES USING DNA DENDRIMERS

In one aspect, the present disclosure provides for a test device fordetecting an analyte in a liquid sample, which device comprises a porousmatrix that comprises a test zone on said porous matrix, said test zonecomprising a test reagent that binds to an analyte or another bindingreagent that binds to said analyte, or is an analyte or an analyteanalog that competes with an analyte in said sample for binding to abinding reagent for said analyte, wherein a liquid sample flowslaterally along said test device and passes (or a liquid sample iscapable of flowing laterally along said test device and passing) saidtest zone to form a detectable signal to indicate presence, absenceand/or amount of said analyte in said liquid sample, the formation ofsaid detectable signal requires the use of a detectable label and a DNAdendrimer, said DNA dendrimer comprises a first component that linkssaid DNA dendrimer to a binding reagent linkable to said DNA dendrimer,an analyte linkable to said DNA dendrimer, or an analyte analog linkableto said DNA dendrimer, and a second component that links said DNAdendrimer to said detectable label linkable to said DNA dendrimer.

In some embodiments, the analyte is not a polynucleotide; or the DNAdendrimer comprises from about 1 to about 324, preferably from about 60to about 324, from about 80 to about 300 or from about 100 to about 200,the first components; or the DNA dendrimer, before the test device isused, is dried on a location on the test device upstream from the testzone. In some embodiments, the test reagent or binding reagentspecifically binds to an analyte.

The first component and the second component can be any suitablesubstances. For example, the first component and/or the second componentcan be an inorganic molecule or moiety, an organic molecule or moiety,or a complex thereof. Exemplary organic molecules or moieties include anamino acid, a peptide, a protein, a nucleoside, a nucleotide, anoligonucleotide, a nucleic acid, a vitamin, a monosaccharide, anoligosaccharide, a carbohydrate, a lipid and a complex thereof.

In some embodiments, the first component and the second component arethe same. In other embodiments, the first component and the secondcomponent are different. In still other embodiments, one of the firstcomponent and the second component is a polynucleotide and the other isa non-polynucleotide moiety. In yet other embodiments, the firstcomponent is a polynucleotide and the second component is anon-polynucleotide moiety, e.g., a polypeptide.

The DNA dendrimer can be linked to any suitable binding reagent via thefirst component. For example, the first component can link the DNAdendrimer to a binding reagent that binds to an analyte, preferably abinding reagent that specifically binds to an analyte. In anotherexample, the first component can link the DNA dendrimer to a bindingreagent that binds to another binding reagent that binds to an analyte,preferably a binding reagent that specifically binds to an analyte.

The DNA dendrimer can be linked to any suitable reagent via the secondcomponent. For example, the second component can link the DNA dendrimerto a detectable label. In another example, the second component can linkthe DNA dendrimer to another binding reagent that is linked to adetectable label.

The present devices can be used in any suitable assay formats, e.g.,sandwich or competitive assay formats. In some embodiments, the testdevice is to be used in a sandwich assay for the analyte and wherein thetest reagent at the test zone binds, and preferably specifically binds,to the analyte, a second binding reagent that binds, and preferablyspecifically binds, to the analyte is used, the second binding reagentcomprises a polynucleotide that is substantially complementary to apolynucleotide that is the first component of a DNA dendrimer. In otherembodiments, the test device is to be used in a sandwich assay for theanalyte and wherein the test reagent at the test zone binds, andpreferably specifically binds, to the analyte, a second binding reagentthat binds to another binding reagent that binds, and preferablyspecifically binds, to an analyte is used, the second binding reagentcomprises a polynucleotide that is substantially complementary to apolynucleotide that is the first component of a DNA dendrimer.

In still other embodiments, the test device is to be used in acompetitive assay for the analyte and wherein the test reagent at thetest zone is an analyte or an analyte analog, a second binding reagentthat binds, and preferably specifically binds, to the analyte is used,the second binding reagent comprises a polynucleotide that issubstantially complementary to a polynucleotide that is the firstcomponent of a DNA dendrimer, and the analyte or an analyte analog atthe test zone competes with an analyte in the sample for binding to thesecond binding reagent. In yet other embodiments, the test device is tobe used in a competitive assay for the analyte and wherein the testreagent at the test zone is an analyte or an analyte analog, a secondbinding reagent that binds to another binding reagent that binds, andpreferably specifically binds, to an analyte is used, the second bindingreagent comprises a polynucleotide that is substantially complementaryto a polynucleotide that is the first component of a DNA dendrimer, andthe analyte or an analyte analog at the test zone competes with ananalyte in the sample for binding to the binding reagent that is boundto the second binding reagent.

The analytes and the various reagents used in connection with thepresent devices, e.g., analyte, analyte analog, test reagent and/orbinding reagent, can be any suitable substances. In some embodiments,the analyte, analyte analog, test reagent and/or binding reagent is aninorganic molecule, an organic molecule or a complex thereof. Exemplaryorganic molecules include an amino acid, a peptide, a protein, anucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a vitamin,a monosaccharide, an oligosaccharide, a carbohydrate, a lipid and acomplex thereof.

In other embodiments, the analyte is a polypeptide, a small molecule oran antigen, and the test reagent and/or binding reagent that binds tothe analyte is an antibody that binds, and preferably specificallybinds, to the polypeptide, small molecule or an antigen. In still otherembodiments, the analyte is a polynucleotide, and the test reagentand/or binding reagent that binds to the analyte is anotherpolynucleotide that is substantially complementary to the analytepolynucleotide. In yet other embodiments, the analyte is apolynucleotide, and the test reagent and/or binding reagent that bindsto the analyte is a non-polynucleotide moiety, e.g., a polypeptide, anantibody or a receptor that binds to the analyte polynucleotide.

The matrix can comprise or be made of any suitable material. In someembodiments, the matrix comprises nitrocellulose, glass fiber,polypropylene, polyethylene (preferably of very high molecular weight),polyvinylidene flouride, ethylene vinylacetate, acrylonitrile and/orpolytetrafluoro-ethylene. See e.g., U.S. Pat. No. 6,187,598. It can beadvantageous to pre-treat the membrane with a surface-active agentduring manufacture, as this can reduce any inherent hydrophobicity inthe membrane and therefore enhance its ability to take up and deliver amoist sample rapidly and efficiently. The matrix can also be made frompaper or other cellulosic materials. In some embodiments, the matrixcomprises or is made of nitrocellulose or glass fiber.

The matrix can also be in any suitable form or shape. In someembodiments, the matrix is in the form a strip or a circle. The matrixcan also comprise or be made of any suitable number of element. In someembodiments, the matrix is a single element or comprises multipleelements.

The present test devices can comprise any suitable additional elements.In some embodiments, the test device can further comprise a sampleapplication element upstream from and in fluid communication with thematrix. In other embodiments, the test device can further comprise aliquid absorption element downstream from and in fluid communicationwith the matrix. In still other embodiments, the test device can furthercomprise a control zone comprising means for indicating proper flow ofthe liquid sample and/or a valid test result. In yet other embodiments,at least a portion of the matrix is supported by a solid backing. In yetother embodiments, the entire matrix is supported by a solid backing.

The various reagents used in connection with the present devices, e.g.,the DNA dendrimer, the various binding reagents and/or the detectable,can be dried on the test devices before use. In some embodiments, asubstance is dried on a portion of the matrix upstream from the testzone, the dried substance being capable of being moved by a liquidsample and/or a further liquid to the test zone and/or a control zone togenerate a detectable signal, the dried substance being at least oneof: 1) the DNA dendrimer; 2) the first binding reagent that binds to theanalyte, the second binding reagent that binds to another bindingreagent that binds to the analyte, the analyte or the analyte analog,each of the first binding reagent, second binding reagent, analyte oranalyte analog being linkable to the DNA dendrimer; and 3) thedetectable label linkable to the DNA dendrimer.

In other embodiments, two substances are dried on a portion of thematrix upstream from the test zone, the dried substances being capableof being moved by a liquid sample and/or a further liquid to the testzone and/or a control zone to generate a detectable signal, the driedsubstances being at least two of: 1) the DNA dendrimer; 2) the firstbinding reagent that binds to the analyte, the second binding reagentthat binds to another binding reagent that binds to the analyte, theanalyte or the analyte analog, each of the first binding reagent, secondbinding reagent, analyte or analyte analog being linkable to the DNAdendrimer; and 3) the label linkable to the DNA dendrimer; the driedsubstances, whether separately or in complex, being capable of beingmoved by a liquid sample and/or a further liquid to the test zone and/ora control zone to generate a detectable signal.

In still other embodiments, three substances are dried on a portion ofthe matrix upstream from the test zone, the dried substances beingcapable of being moved by a liquid sample and/or a further liquid to thetest zone and/or a control zone to generate a detectable signal, thedried substances being all of: 1) the DNA dendrimer; 2) the firstbinding reagent that binds to the analyte, the second binding reagentthat binds to another binding reagent that binds to the analyte, theanalyte or the analyte analog, each of the first binding reagent, secondbinding reagent, analyte or analyte analog being linkable to the DNAdendrimer; and 3) the label linkable to the DNA dendrimer.

The above substance(s) can be dried on any suitable location of the testdevices. In some embodiments, the substance(s) is dried on a conjugateelement that is upstream from the test zone. In other embodiments, thesubstance(s) is located downstream from a sample application place onthe test device. In still other embodiments, the substance(s) is locatedupstream from a sample application place on the test device.

Any suitable detectable label can be used in connection with the presenttest devices. In some embodiments, the detectable label is a solublelabel, e.g., a soluble enzyme or fluorescent label. In otherembodiments, the detectable label is a particle label. The particlelabel can be a visible or a non-visible particle label. In someembodiments, the visible particle label is selected from the groupconsisting of a gold particle, a latex particle, a Q-Dot, a carbonnanotube, a silver particle and a silver coated particle. In someembodiments, the non-visible particle label is a fluorescent particle. Adetectable label can also be a moiety that effects a change in massand/or charge. A DNA dendrimer can effect a change in mass and/orcharge. In some embodiments, a detectable label can be a DNA dendrimer,e.g., the DNA dendrimer itself or an additional DNA dendrimer.

In some embodiments, the substance(s) is dried in the presence of amaterial that: a) stabilizes the dried substance(s); b) facilitatessolubilization or resuspension of the dried substance(s) in a liquid;and/or c) facilitates mobility of the dried substance(s). Any suitablematerial can be used. For example, the material can be a protein, apeptide, a polysaccharide, a sugar, a polymer, a gelatin and adetergent. See e.g., U.S. Pat. Nos. 5,120,643 and 6,187,598.

In some embodiments, a sample liquid alone is used to transport theanalyte and/or the substance(s) to the test zone. In other embodiments,a developing liquid is used to transport the analyte and/or thesubstance(s) to the test zone.

The test device can further comprise a housing that covers at least aportion of the test device, wherein the housing comprises a sampleapplication port to allow sample application upstream from or to thetest zone and an optic opening around the test zone to allow signaldetection at the test zone and/or the control zone. In some embodiments,the housing covers the entire test device. In other embodiments, atleast a portion of the sample receiving portion of the matrix or thesample application element is not covered by the housing and a sample isapplied to the portion of the sample receiving portion of the matrix orthe sample application element outside the housing and then transportedto the test zone.

The DNA dendrimer, the various binding reagents and the detectable labelcan be linked in any suitable manner, e.g., non-covalently linked orcovalently linked. In some embodiments, none of: 1) the DNA dendrimer;2) the first binding reagent that binds to the analyte, the secondbinding reagent that binds to another binding reagent that binds to theanalyte, the analyte or the analyte analog, each of the first bindingreagent, second binding reagent, analyte or analyte analog beinglinkable to the DNA dendrimer; and 3) the detectable label linkable tothe DNA dendrimer; is covalently bound or crosslinked to each other.

In other embodiments, at least two of: 1) the DNA dendrimer; 2) thefirst binding reagent that binds to the analyte, the second bindingreagent that binds to another binding reagent that binds to the analyte,the analyte or the analyte analog, each of the first binding reagent,second binding reagent, analyte or analyte analog being linkable to theDNA dendrimer; and 3) the detectable label linkable to the DNAdendrimer; are covalently bound or crosslinked to each other.

In still other embodiments, all of: 1) the DNA dendrimer; 2) the firstbinding reagent that binds to the analyte, the second binding reagentthat binds to another binding reagent that binds to the analyte, theanalyte or the analyte analog, each of the first binding reagent, secondbinding reagent, analyte or analyte analog being linkable to the DNAdendrimer; and 3) the detectable label linkable to the DNA dendrimer;are covalently bound or crosslinked to each other.

Any suitable DNA dendrimer can be used. For example, the DNA dendrimersdisclosed and/or claimed in the U.S. Pat. Nos. 5,175,270, 5,487,973,6,046,038, 6,072,043, 6,110,687, 6,117,631 and 6,274,723 can be used.The DNA dendrimers can comprise any suitable number of second componentsthat link the DNA dendrimer to a detectable label. In some embodiments,the DNA dendrimer comprises from about 10 to about 1,500, preferablyfrom about 40 to about 1,500, from about 100 to about 1,400, from about500 to about 1,200, or from about 900 to about 1,100, second componentsthat link the DNA dendrimer to a detectable label linkable to the DNAdendrimer. The label can be the same or can be different.

The DNA dendrimers can comprise any suitable number of DNA nucleotides.In some embodiments, the DNA dendrimer comprises from about 400 to about80,000, preferably from about 4,000 to about 80,000, from about 10,000to about 80,000, from about 30,000 to about 80,000, or from about 60,000to about 80,000, DNA nucleotides.

The DNA dendrimers can comprise any suitable number of layers. In someembodiments, the DNA dendrimer comprises a one-layer, a two-layer, athree-layer or a four-layer structure.

In some embodiments, the present disclosure provides for a test devicefor detecting an analyte in a liquid sample wherein the liquid samplehas moved laterally along the test device to generate a detectablesignal at the test zone.

C. METHODS FOR DETECTING AN ANALYTE IN A LIQUID SAMPLE

In another aspect, the present disclosure provides for a method fordetecting an analyte in a liquid sample, which method comprises: a)contacting a liquid sample with the test device described in the aboveSection B, wherein the liquid sample is applied to a site of the testdevice upstream of the test zone; b) transporting an analyte, if presentin the liquid sample, a detectable label and a DNA dendrimer to the testzone; and c) assessing the presence, absence, and/or amount of a signalgenerated by the detectable label at the test zone to determine thepresence, absence and/or amount of the analyte in the liquid sample.

The present methods can be used in any suitable assay formats. Forexample, the DNA dendrimer, the various binding reagents and/or thedetectable label can be premixed with a sample liquid and/or developingliquid, and then the mixture is applied to the test device to initiatean assay. Alternatively, one or more of the DNA dendrimer, the variousbinding reagents and/or the detectable label can be dried on a suitablelocation of the test device, and a sample liquid and/or developingliquid is applied to the test device to initiate an assay.

In some embodiments, the liquid sample and at least one of: 1) the DNAdendrimer; 2) the first binding reagent that binds to the analyte, thesecond binding reagent that binds to another binding reagent that bindsto the analyte, the analyte or the analyte analog, each of the firstbinding reagent, second binding reagent, analyte or analyte analog beinglinkable to the DNA dendrimer; and 3) the detectable label linkable tothe DNA dendrimer; are premixed to form a mixture and the mixture isapplied to the test device.

In other embodiments, the liquid sample and at least two of: 1) the DNAdendrimer; 2) the first binding reagent that binds to the analyte, thesecond binding reagent that binds to another binding reagent that bindsto the analyte, the analyte or the analyte analog, each of the firstbinding reagent, second binding reagent, analyte or analyte analog beinglinkable to the DNA dendrimer; and 3) the detectable label linkable tothe DNA dendrimer; are premixed to form a mixture and the mixture isapplied to the test device.

In still other embodiments, the liquid sample and all of: 1) the DNAdendrimer; 2) the first binding reagent that binds to the analyte, thesecond binding reagent that binds to another binding reagent that bindsto the analyte, the analyte or the analyte analog, each of the firstbinding reagent, second binding reagent, analyte or analyte analog beinglinkable to the DNA dendrimer; and 3) the detectable label linkable tothe DNA dendrimer; are premixed to form a mixture and the mixture isapplied to the test device.

The present methods can be conducted in a liquid comprising a surfactantor detergent, e.g., Tween-20. In some embodiments, the method isconducted in a liquid comprising from about 0.001% (v/v) to about 5%(v/v), preferably, from about 0.01% (v/v) to about 0.5% (v/v), or atabout 0.01% (v/v) or less Tween-20. The present methods can also beconducted in a liquid comprising a polymer, e.g., dextran sulfate. Insome embodiments, the method is conducted in a liquid comprising fromabout 0.1% (v/v) to about 5% (v/v), preferably from about 0.5% (v/v) toabout 1% (v/v), dextran sulfate.

In some embodiments, a substance is dried on a portion of the testdevice upstream from the test zone, the dried substance is solubilizedor resuspended, and transported to the test zone and/or a control zoneto generate a detectable signal, the dried substance being at least oneof: 1) the DNA dendrimer; 2) the first binding reagent that binds to theanalyte, the second binding reagent that binds to another bindingreagent that binds to the analyte, the analyte or the analyte analog,each of the first binding reagent, second binding reagent, analyte oranalyte analog being linkable to the DNA dendrimer; and 3) thedetectable label linkable to the DNA dendrimer.

The substance can be dried on any suitable location of the test device.In some embodiments, the dried substance is located downstream from thesample application site, and the dried substance is solubilized orresuspended, and transported to the test zone and/or a control zone bythe liquid sample. In other embodiments, the dried substance is locatedupstream from the sample application site, and the dried substance issolubilized or resuspended, and transported to the test zone and/or acontrol zone by another liquid. In still other embodiments, the driedsubstance is solubilized or resuspended, and transported to the testzone and/or a control zone by the liquid sample alone. In yet otherembodiments, the analyte and/or dried substance is solubilized orresuspended, and transported to the test zone and/or a control zone byanother liquid.

The present methods can be used to assess an analyte in any suitablesample. In some embodiments, the present methods can be used to assessan analyte in a body fluid sample, e.g., a whole blood, a serum, aplasma and a urine sample. In other embodiments, the present methods canbe used to assess an analyte in a sample derived from a biological, aforensics, a food, a biowarfare, or an environmental source.

The present methods can be used for any suitable purpose. In someembodiments, the present methods can be used to quantify orsemi-quantify the amount of an analyte in a liquid sample. In otherembodiments, the present methods can be used to detect multiple analytesin a liquid sample. In still other embodiments, the present methods canbe used to quantify or semi-quantify the amounts of the multipleanalytes in the liquid sample.

The present methods can be used to assess any suitable analyte. In someembodiments, the present methods can be used to assess an analyteselected from the group consisting of a cell, a virus and a molecule.

In some embodiments, the present methods are automated and/or are usedin high throughput format.

D. LATERAL ASSAY DEVICES USING DNA DENDRIMER LINKED TO A DETECTABLELABEL NON-COVALENTLY

In still another aspect, the present disclosure provides for a testdevice for detecting an analyte in a liquid sample, which devicecomprises a porous matrix that comprises a test zone on said porousmatrix, said test zone comprising a test reagent that binds to ananalyte or another binding reagent that binds to said analyte, or is ananalyte or an analyte analog that competes with an analyte in saidsample for binding to a binding reagent for said analyte, wherein aliquid sample flows laterally along said test device and passes (or aliquid sample is capable of flowing laterally along said test device andpassing) said test zone to form a detectable signal to indicatepresence, absence and/or amount of said analyte in said liquid sample,the formation of said detectable signal requires the use of a DNAdendrimer linked to a detectable label covalently or non-covalently, andsaid DNA dendrimer comprises a component that links said DNA dendrimerto a binding reagent linkable to said DNA dendrimer, an analyte linkableto said DNA dendrimer, or an analyte analog linkable to said DNAdendrimer.

In some embodiments, the analyte is not a polynucleotide; or the DNAdendrimer comprises from about 1 to about 324, preferably from about 60to about 324, from about 80 to about 300 or from about 100 to about 200,the first components; or the DNA dendrimer, before the test device isused, is dried on a location on the test device upstream from the testzone. In some embodiments, the test reagent or binding reagentspecifically binds to an analyte.

The component can be any suitable substance. For example, the componentcan be an inorganic molecule or moiety, an organic molecule or moiety,or a complex thereof. Exemplary organic molecules or moieties include anamino acid, a peptide, a protein, a nucleoside, a nucleotide, anoligonucleotide, a nucleic acid, a vitamin, a monosaccharide, anoligosaccharide, a carbohydrate, a lipid and a complex thereof. In someembodiments, the component is a polynucleotide.

The present devices can be used in any suitable assay formats, e.g.,sandwich or competitive assay formats. In some embodiments, the testdevice is to be used in a sandwich assay for the analyte and wherein thetest reagent at the test zone binds, preferably specifically binds, tothe analyte, a second binding reagent that binds, preferablyspecifically binds, to the analyte is used, the second binding reagentcomprises a polynucleotide that is substantially complementary to apolynucleotide that is the component of a DNA dendrimer. In otherembodiments, the test device is to be used in a sandwich assay for theanalyte and wherein the test reagent at the test zone binds, preferablyspecifically binds, to the analyte, a second binding reagent that bindsto another binding reagent that binds, preferably specifically binds, toan analyte is used, the second binding reagent comprises apolynucleotide that is substantially complementary to a polynucleotidethat is the component of a DNA dendrimer.

In still other embodiments, the test device is to be used in acompetitive assay for the analyte and wherein the test reagent at thetest zone is an analyte or an analyte analog, a second binding reagentthat binds, preferably specifically binds, to the analyte is used, thesecond binding reagent comprises a polynucleotide that is substantiallycomplementary to a polynucleotide that is the component of a DNAdendrimer, and the analyte or an analyte analog at the test zonecompetes with an analyte in the sample for binding to the second bindingreagent. In yet other embodiments, the test device is to be used in acompetitive assay for the analyte and wherein the test reagent at thetest zone is an analyte or an analyte analog, a second binding reagentthat binds to another binding reagent that binds, preferablyspecifically binds, to an analyte is used, the second binding reagentcomprises a polynucleotide that is substantially complementary to apolynucleotide that is the component of a DNA dendrimer, and the analyteor an analyte analog at the test zone competes with an analyte in thesample for binding to the binding reagent that is bound to the secondbinding reagent.

The analytes and the various reagents used in connection with thepresent devices, e.g., analyte, analyte analog, test reagent and/orbinding reagent, can be any suitable substances. In some embodiments,the analyte, analyte analog, test reagent and/or binding reagent is aninorganic molecule, an organic molecule or a complex thereof. Exemplaryorganic molecule can be an amino acid, a peptide, a protein, anucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a vitamin,a monosaccharide, an oligosaccharide, a carbohydrate, a lipid and acomplex thereof.

In other embodiments, the analyte is a polypeptide, a small molecule oran antigen, and the test reagent and/or binding reagent that binds tothe analyte is an antibody that binds, preferably specifically binds, tothe polypeptide, small molecule or antigen. In still other embodiments,the analyte is a polynucleotide, and the test reagent and/or bindingreagent that binds to the analyte is another polynucleotide that issubstantially complementary to the analyte polynucleotide. In yet otherembodiments, the analyte is a polynucleotide, and the test reagentand/or binding reagent that binds to the analyte is a non-polynucleotidemoiety, e.g., a polypeptide, an antibody or a receptor that binds to theanalyte polynucleotide.

The matrix can comprise or be made of any suitable material. In someembodiments, the matrix comprises nitrocellulose, glass fiber,polypropylene, polyethylene (preferably of very high molecular weight),polyvinylidene flouride, ethylene vinylacetate, acrylonitrile and/orpolytetrafluoro-ethylene. See e.g., U.S. Pat. No. 6,187,598. It can beadvantageous to pre-treat the membrane with a surface-active agentduring manufacture, as this can reduce any inherent hydrophobicity inthe membrane and therefore enhance its ability to take up and deliver amoist sample rapidly and efficiently. The matrix can also be made frompaper or other cellulosic materials. In some embodiments, the matrixcomprises or is made of nitrocellulose or glass fiber.

The matrix can also be in any suitable form or shape. In someembodiments, the matrix is in the form a strip or a circle. The matrixcan also comprise or be made of any suitable number of element. In someembodiments, the matrix is a single element or comprises multipleelements.

The present test devices can comprise any suitable additional elements.In some embodiments, the test device can further comprise a sampleapplication element upstream from and in fluid communication with thematrix. In other embodiments, the test device can further comprise aliquid absorption element downstream from and in fluid communicationwith the matrix. In still other embodiments, the test device can furthercomprise a control zone comprising means for indicating proper flow ofthe liquid sample and/or a valid test result. In yet other embodiments,at least a portion of the matrix is supported by a solid backing. In yetother embodiments, the entire matrix is supported by a solid backing.

The various reagents used in connection with the present devices, e.g.,the DNA dendrimer and/or the various binding reagents, can be dried onthe test devices before use. In some embodiments, a substance is driedon a portion of the matrix upstream from the test zone, the driedsubstance being capable of being moved by a liquid sample and/or afurther liquid to the test zone and/or a control zone to generate adetectable signal, the dried substance being at least one of: 1) the DNAdendrimer; and 2) the first binding reagent that binds to the analyte,the second binding reagent that binds to another binding reagent thatbinds to the analyte, the analyte or the analyte analog, each of thefirst binding reagent, second binding reagent, analyte or analyte analogbeing linkable to the DNA dendrimer. In other embodiments, both of theDNA dendrimer, and the first binding reagent that binds to the analyte,the second binding reagent that binds to another binding reagent thatbinds to the analyte, the analyte or the analyte analog, are dried on aportion of the matrix upstream from the test zone.

The substance(s) can be dried on any suitable location of the testdevice. In some embodiments, the substance(s) is dried on a conjugateelement that is upstream from the test zone. In other embodiments, thesubstance(s) is located downstream from a sample application place onthe test device. In still other embodiments, the substance(s) is locatedupstream from a sample application place on the test device.

Any suitable detectable label can be used in connection with the presenttest devices. In some embodiments, the detectable label is a solublelabel, e.g., a soluble enzyme or fluorescent label. In otherembodiments, the detectable label is a particle label. The particlelabel can be a visible or a non-visible particle label. In someembodiments, the visible particle label is selected from the groupconsisting of a gold particle, a latex particle, a Q-Dot, a carbonnanotube, a silver particle and a silver coated particle. In someembodiments, the non-visible particle label is a fluorescent particle. Adetectable label can also be a moiety that effects a change in massand/or charge. A DNA dendrimer can effect a change in mass and/orcharge. In some embodiments, a detectable label can be a DNA dendrimer,e.g., the DNA dendrimer itself or an additional DNA dendrimer.

In some embodiments, the substance(s) is dried in the presence of amaterial that: a) stabilizes the dried substance(s); b) facilitatessolubilization or resuspension of the dried substance(s) in a liquid;and/or c) facilitates mobility of the dried substance(s). Any suitablematerial can be used. For example, the material can be a protein, apeptide, a polysaccharide, a sugar, a polymer, a gelatin and adetergent. See e.g., U.S. Pat. Nos. 5,120,643 and 6,187,598.

In some embodiments, a sample liquid alone is used to transport theanalyte and/or the substance(s) to the test zone. In other embodiments,a developing liquid is used to transport the analyte and/or thesubstance(s) to the test zone.

The test device can further comprise a housing. In some embodiments, thetest device further comprise a housing that covers at least a portion ofthe test device, wherein the housing comprises a sample application portto allow sample application upstream from or to the test zone and anoptic opening around the test zone to allow signal detection at the testzone and/or the control zone. In other embodiments, the housing coversthe entire test device. In still other embodiments, at least a portionof the sample receiving portion of the matrix or the sample applicationelement is not covered by the housing and a sample is applied to theportion of the sample receiving portion of the matrix or the sampleapplication element outside the housing and then transported to the testzone.

Any suitable DNA dendrimer can be used. For example, the DNA dendrimersdisclosed and/or claimed in the U.S. Pat. Nos. 5,175,270, 5,487,973,6,046,038, 6,072,043, 6,110,687, 6,117,631 and 6,274,723 can be used.The DNA dendrimers can comprise any suitable number of components thatlink the DNA dendrimer to a binding reagent linkable to said DNAdendrimer. In some embodiments, the DNA dendrimer comprises from about 1to about 324, preferably from about 60 to about 324, from about 80 toabout 300 or from about 100 to about 200, components that link the DNAdendrimer to a binding reagent linkable to said DNA dendrimer.

The DNA dendrimers can comprise any suitable number of the detectablelabel. In some embodiments, the DNA dendrimers comprises from about 10to about 1,500, preferably from about 40 to about 1,500, or from about100 to about 1,200, or from about 900 to about 1,100, the detectablelabels. The label can be the same or can be different.

The DNA dendrimers can comprise any suitable number of DNA nucleotides.In some embodiments, the DNA dendrimer comprises from about 400 to about80,000, preferably from about 4,000 to about 80,000, from about 10,000to about 80,000, from about 30,000 to about 80,000, or from about 60,000to about 80,000, DNA nucleotides.

The DNA dendrimers can comprise any suitable number of layers. In someembodiments, the DNA dendrimer comprises a one-layer, a two-layer, athree-layer or a four-layer structure.

In some embodiments, the present disclosure provides for a test devicewherein the liquid sample has moved laterally along the test device togenerate a detectable signal at the test zone.

E. METHODS FOR DETECTING AN ANALYTE IN A LIQUID SAMPLE

In yet another aspect, the present disclosure provides for a method fordetecting an analyte in a liquid sample, which method comprises: a)contacting a liquid sample with the test device described in the aboveSection D, wherein the liquid sample is applied to a site of the testdevice upstream of the test zone; b) transporting an analyte, if presentin the liquid sample, a detectable label and a DNA dendrimer to the testzone; and c) assessing the presence, absence, and/or amount of a signalgenerated by the detectable label at the test zone to determine thepresence, absence and/or amount of the analyte in the liquid sample.

The present methods can be used in any suitable assay formats. Forexample, the DNA dendrimer and/or the various binding reagents can bepremixed with a sample liquid and/or developing liquid, and then themixture is applied to the test device to initiate an assay.Alternatively, the DNA dendrimer and/or the various binding reagents canbe dried on a suitable location of the test device, and a sample liquidand/or developing liquid is applied to the test device to initiate anassay.

In some embodiments, the liquid sample and at least one of: 1) the DNAdendrimer linked to a detectable label non-covalently; and 2) the firstbinding reagent that binds to the analyte, the second binding reagentthat binds to another binding reagent that binds to the analyte, theanalyte or the analyte analog, each of the first binding reagent, secondbinding reagent, analyte or analyte analog being linkable to the DNAdendrimer; are premixed to form a mixture and the mixture is applied tothe test device.

In other embodiments, the liquid sample and both of: 1) the DNAdendrimer linked to a detectable label non-covalently; and 2) the firstbinding reagent that binds to the analyte, the second binding reagentthat binds to another binding reagent that binds to the analyte, theanalyte or the analyte analog, each of the first binding reagent, secondbinding reagent, analyte or analyte analog being linkable to the DNAdendrimer; are premixed to form a mixture and the mixture is applied tothe test device.

The present methods can be conducted in a liquid comprising a surfactantor detergent, e.g., Tween-20. In some embodiments, the method isconducted in a liquid comprising from about 0.001% (v/v) to about 5%(v/v), preferably, from about 0.01% (v/v) to about 0.5% (v/v), or atabout 0.01% (v/v) or less Tween-20. The present methods can also beconducted in a liquid comprising a polymer, e.g., dextran sulfate. Insome embodiments, the method is conducted in a liquid comprising fromabout 0.1% (v/v) to about 5% (v/v), preferably from about 0.5% (v/v) toabout 1% (v/v), dextran sulfate.

In some embodiments, a substance is dried on a portion of the testdevice upstream from the test zone, the dried substance is capable ofbeing moved by a liquid sample and/or a further liquid to the test zoneand/or a control zone to generate a detectable signal, the driedsubstance being at least one of: 1) the DNA dendrimer linked to adetectable label non-covalently; and 2) the first binding reagent thatbinds to the analyte, the second binding reagent that binds to anotherbinding reagent that binds to the analyte, the analyte or the analyteanalog, each of the first binding reagent, second binding reagent,analyte or analyte analog being linkable to the DNA dendrimer.

The substance can be dried on any suitable location of the test device.In some embodiments, the dried substance is located downstream from thesample application site, and the dried substance is solubilized orresuspended, and transported to the test zone and/or a control zone bythe liquid sample. In other embodiments, the dried substance is locatedupstream from the sample application site, and the dried substance issolubilized or resuspended, and transported to the test zone and/or acontrol zone by another liquid. In still other embodiments, the driedsubstance is solubilized or resuspended, and transported to the testzone and/or a control zone by the liquid sample alone. In yet otherembodiments, the analyte and/or dried substance is solubilized orresuspended, and transported to the test zone and/or a control zone byanother liquid.

The present methods can be used to assess an analyte in any suitablesample. In some embodiments, the present methods can be used to assessan analyte in a body fluid sample, e.g., a whole blood, a serum, aplasma and a urine sample. In other embodiments, the present methods canbe used to assess an analyte in a sample derived from a biological, aforensics, a food, a biowarfare, or an environmental source.

The present methods can be used for any suitable purpose. In someembodiments, the present methods can be used to quantify orsemi-quantify the amount of an analyte in a liquid sample. In otherembodiments, the present methods can be used to detect multiple analytesin a liquid sample. In still other embodiments, the present methods canbe used to quantify or semi-quantify the amounts of the multipleanalytes in the liquid sample.

The present methods can be used to assess any suitable analyte. In someembodiments, the present methods can be used to assess an analyteselected from the group consisting of a cell, a virus and a molecule.

In some embodiments, the present methods are automated and/or are usedin high throughput format.

F. EXEMPLARY EMBODIMENTS

In exemplary embodiments, this disclosure describes the use of DNAdendrimers as signal amplification devices in “point-of-care” (POC)detection assays, typified as immunoassays that require little skill bythe end-user, provide simple readouts and results interpretation, andare performed in a relatively short period of time with minimalprocedural steps. Typically, POC tests include a variety of assayformats utilizing a variety of different signaling strategiesincorporating visible and non-visible signaling devices, includingseveral types of immunochromatographic assays (visible readout lateralflow, dual path platform, etc.), fluorescent, electrochemical andenzymatic detection assays using membranes and lateral flow typedevices, and assays which incorporate the use of microfluidics andbiosensors (with or without membranes or paper substrates). These assaysmay be capable of detecting a different single analyte or multipleanalytes simultaneously. Commonly, POC tests are mostly represented bylateral flow immunoassays, which usually include a membrane strip whichcontain an immobilized ligand (antibody or antigen) in a “test” line onthe membrane which is capable of binding to the analyte of interest andindicates the presence of the analyte in a test sample when the testline is positive. Lateral flow assays also often contain a “control”line which indicates that the assay has worked appropriately. Othercomponents of a lateral flow assay include a sample pad where theanalyte containing sample is added to the strip assay, a conjugate padwhich contains dried down assay reagents, a membrane (typicallynitrocellulose) capable of capillary action and the site of theimmunological reaction between the immobilized ligand, the analyte andthe signal generating ligand and related devices, and a wicking pad onthe distal end of the membrane which serves to draw liquid via capillaryaction in the membrane from the sample pad, through the membrane, andinto the wicking pad. When liquid sample is added to the sample pad, theliquid flows from the sample pad into the conjugate pad, where anysignal generating reagents previously dried into the conjugate pad(e.g., gold particle conjugated with anti-analyte antibody) arere-hydrated and are carried with the sample liquid front into themembrane. The analyte and signal generating reagents then react with thetest line (T) and control line (C), and a positive result of detectionof the analyte is indicated by a signal at the test line. The wick orabsorbent pad at the distal end of the membrane serves to “pull” theliquid through the membrane via capillary actions (see FIG. 1).

The use of DNA dendrimers in lateral flow and other POC assays has onlyrecently been explored (see e.g., U.S. patent application US2011/0160090 A1), even though these applications were similar in conceptto other immunoassays already incorporating DNA dendrimers as signalamplifiers (e.g., ELISA). Previously, it was assumed by the inventorsthat DNA dendrimers would likely not be suitable for use in lateral flowassays given the nature and size of the dendrimers and prior experiencesof poor performance of the dendrimers in other membrane based assays(e.g. Western blot immunoassays), which included low signalamplification and very high non-specific background. Notwithstandingthis assumption, and given the ability of the DNA dendrimer to deliverhundreds of label moieties to a single binding site (compared to asingle or at most 5-10 labels using non-dendrimer lateral flowreagents), we decided to perform lateral flow assays utilizing DNAdendrimers as signal amplifiers. Specifically, DNA dendrimers containingnearly 1,000 biotin moieties per dendrimer were synthesized and testedin a model lateral flow hCG assay, with an anti-hCGantibody-oligonucleotide conjugate serving as the “bridge” reagentbetween the hCG analyte and a streptavidin-gold conjugate serving as thesignal generator (the streptavidin-gold binding the biotins on thedendrimers). A standard hCG assay utilizing gold particles conjugatedwith anti-hCG antibody was also performed (FIG. 2). To our surprise, thelateral flow assay with the DNA dendrimers performed extremely well,providing up to 128 fold of improvement of sensitivity over the standardhCG assay, with little or no non-specific background signal across theassay membrane. Note it was not expected that DNA dendrimers wouldperform well in lateral flow assays as we expected the dendrimers wouldnot be able to efficiently perfuse the membrane during the performanceof a lateral flow assay due to the size and highly branched structure ofthe dendrimer. Further, the non-specific binding of signal generatingmoieties in DNA dendrimer lateral flow assays does occur but iscontrollable via blocking and buffer selection activities.

Specific Applications of DNA Dendrimers in Lateral Flow POC Assays

DNA dendrimers can be used in POC lateral flow protein detection assaysin sandwich and/or competitive assay formats. In some embodiments, theDNA dendrimers are used in a direct sandwich assay. This assay formatrequires the immobilization of a first antibody specific for theanalyte, typically as an antibody stripe line located on a membranestrip capable of drawing liquid from one end to another of the membranevia capillary action. An aqueous sample containing the analyte is addedto the end of membrane strip and the analyte is captured by theimmobilized first antibody. A second antibody specific for the analyte(but directed to a different epitope than the first immobilizedantibody) and conjugated to an oligonucleotide (the “antibody-oligoconjugate”) is allowed to bind to the analyte. The antibody-oligoconjugate is either separate from or previously bound to a DNA dendrimercontaining labeling moieties capable of generating signal or bindingsignaling devices. The DNA dendrimer is bound to the antibody-oligoconjugate and generates signal located on the test line located on themembrane strip. Visible signal is typically generated by binding goldparticles to the label moieties on the DNA dendrimer (e.g.streptavidin-gold particles binding to DNA dendrimer-bound biotinmoieties) or by label moieties bound to the DNA dendrimer capable ofgenerating signal directly (e.g. fluorescent dyes). An example of thismethod would include an antibody-oligo conjugate that contains anantibody that binds directly to an analyte such as human chorionicgonadotropin (hCG), thereby forming a direct link between theantibody-oligo conjugate and the immobilized analyte. Generally, thehigher the signal, the more positive the result.

In some embodiments, the DNA dendrimers are used in an indirect sandwichassay. This format is very similar to the “direct sandwich assay” exceptthat the antibody-oligo conjugate contains an antibody that does notbind directly to the analyte, but rather binds to another component thatin turns binds to the analyte. An example of this method would includean antibody-oligo conjugate that contains an antibody that binds toanother antibody which in turn binds directly to an analyte such ashuman chorionic gonadotropin (hCG), thereby forming an indirect linkbetween the antibody-oligo conjugate and the immobilized analyte.Generally, the higher the signal, the more positive the result.

In some embodiments, the DNA dendrimers are used in a direct competitiveassay. This assay format generally incorporates the use of immobilizedanalyte (rather than a first antibody) which then binds to anantibody-oligo conjugate, a DNA dendrimer and a label generating moiety.When analyte-containing sample is added, soluble antibody-oligoconjugate is bound to soluble analyte, which competes for the binding ofthe antibody-oligo conjugate to the immobilized analyte, thereby causinga result of lower signal (or no signal). An example of this method wouldinclude an antibody-oligo conjugate that contains an antibody that bindsdirectly to an analyte such as human chorionic gonadotropin (hCG),thereby forming a direct link between the antibody-oligo conjugate andthe soluble analyte, and which competes for the binding of theantibody-oligo conjugate, DNA dendrimer and signal generating moietiesto the immobilized hCG analyte. Generally, the lower the signal, themore positive the result.

In some embodiments, the DNA dendrimers are used in an indirectcompetitive assay. This format is very similar to the “directcompetitive assay” except that the antibody-oligo conjugate contains anantibody that does not bind directly to the analyte, but rather binds toanother component that in turns binds to the analyte. An example of thismethod would include an antibody-oligo conjugate that contains anantibody that binds to another antibody which in turn binds directly toan analyte such as human chorionic gonadotropin (hCG), thereby formingan indirect link between the antibody-oligo conjugate and the solublehCG analyte. Generally, the lower the signal, the more positive theresult.

Further, DNA dendrimers are used in POC nucleic acid detection assays.In some embodiments, the DNA dendrimers are used in a direct detectionof immobilized nucleic acid target. This assay would include theimmobilization of a nucleic acid probe onto a POC device, which in turnwould hybridize to a first nucleic acid sequence located in a nucleicacid molecule of interest in a sample, which in turn would be directlybound by a DNA dendrimer containing a nucleic acid probe targetingdevice capable of hybridizing to a second nucleic acid sequence locatedin the aforementioned nucleic acid molecule of interest in a sample.Signal may be generated in this assay as previously described for theprotein detection assays. An example of this method would include animmobilized DNA probe specific for a cystic fibrosis (CF) mutationhybridizing to the specifically PCR amplified DNA amplicon containingthe partial gene sequence for the CF gene and mutation of interest,further hybridized to the DNA dendrimer-bound DNA oligonucleotidespecific for the CF gene (but to another portion of the sequence whichwill not interfere with the binding of the immobilized probe), therebyforming a direct link between the DNA dendrimer and the immobilizedhybridized CF amplicon. Generally, the higher the signal, the morepositive the result.

In some embodiments, the DNA dendrimers are used in an indirectdetection of immobilized target. This assay is similar to the “directdetection of immobilized nucleic acid target”, except that the DNAdendrimer is indirectly bound to the immobilized nucleic acid moleculeof interest. Indirect binding may be via a nucleic acid molecule, or viaother means, such as binding to a hapten contained within a device inturn bound to the immobilized nucleic acid molecule of interest. Anexample of this method would include an immobilized DNA probe specificfor a cystic fibrosis (CF) mutation hybridizing to the specifically PCRamplified DNA amplicon containing the partial gene sequence for the CFgene and mutation of interest, further hybridized to a second solubleDNA probe specific for another portion of the CF amplicon and containinga biotin on its 5 prime (5′) end, and a DNA dendrimer containing ananti-biotin antibody-oligo conjugate, thereby forming an indirect linkbetween the DNA dendrimer and the immobilized hybridized CF amplicon.Generally, the higher the signal, the more positive the result.

In some embodiments, the DNA dendrimers are used in a competitivenucleic acid POC assays. As discussed above for protein assays,competitive POC nucleic acid detection assays, both direct and indirect,are possible, based on the premise that the labeled moiety (DNAdendrimer or labeled device bound to DNA dendrimer) will bind to solubleanalyte and will compete for the binding to immobilized ligand.Generally, the lower the signal, the more positive the result.

In exemplary embodiments, several novel aspects relate to the use of theDNA dendrimer in the above POC assays. In some embodiments, theimprovement of sensitivity when using DNA dendrimers in POC assays wasnot directly proportional to the number of label moieties bound to thedendrimer, with the sensitivity improvement less than would otherwise bepredicted and in a non-linear manner when increasing the number of labelmoieties from 240 to 480 to 960 labels per dendrimer, while keeping thenumber of targeting devices constant. Further, there was no improvementor a slight loss of sensitivity when using a dendrimer with 1,440 labelsper dendrimer. Conversely, sensitivity was significantly improved byincreasing the number of targeting devices on the dendrimer, which webelieve improved the probability of binding (or collision) between theDNA dendrimer and the targeted analyte, either via direct or indirectbinding between said DNA dendrimer and analyte.

In some embodiments, the DNA dendrimers (with and/or without relatedassay reagents) are “dried down” onto the POC test during themanufacture of the POC test components, thereby reducing the number ofsteps required to perform the POC assay. Typically, assay reagents,including antibody-gold particle conjugates, are dried down onto aconjugate pad for POC lateral flow test strips to reduce the number ofsteps required to perform the assay. The dried down reagent(s) arerehydrated via the addition of the aqueous test sample or by theaddition of aqueous buffers added to the dehydrated reagent(s) duringthe assay procedure, eliminating the need to add these reagents asaqueous preparations during the performance of the assay. To theinventors' knowledge, DNA dendrimers and antibody-oligo conjugates hadnot been dried down in any assay format, and it was unknown whetherdried dendrimers and antibody-oligo conjugates would performappropriately after rehydration. We have clearly demonstrated that DNAdendrimer and antibody-oligo conjugates can be dried down onto a POClateral flow test strip using manufacturing methods similar to thoseused for other lateral flow reagents, and the dendrimer andantibody-oligo conjugates perform as well as or better than reagentsused in a non-dried down liquid state.

In some embodiments, the ability to vary the number of steps in a POCassay using dried down DNA dendrimers is combined with some or all ofthe assay components. Prior POC lateral flow methods include one-stepand multi-step assays which incorporate reagents that are either alldried down onto the lateral flow test strip, or contain some reagentswhich are dried down and others which are not dried down but are used asa liquid reagent during the assay. These reagents include but are notlimited to the DNA dendrimer, antibody-oligo conjugate, gold particleconjugate capable of binding to the DNA dendrimer, native antibodyspecific for the analyte (for an indirect lateral flow assay), and otherbuffers and blockers as required for performing the assay. We have alsofound that the DNA dendrimer may be dried down onto the lateral flowtest strip in combination with all the other reagent components requiredfor the test, resulting in a one-step process. Further, we have foundthat one or more of the aforementioned reagent components may be usedsingly or in combination as components in a POC lateral flow assay,resulting in a method that may require two, three or more steps toperform.

In some embodiments, DNA dendrimers are used with ancillary labeledparticles, including gold and latex particles, and various bridgingstrategies are utilized for binding signaling particles to thedendrimers. DNA dendrimers have been adapted for use with various typesof particles that impart a visible or non-visible signal to POC assays.For visible POC lateral flow assays, the use of gold particles capableof binding to moieties incorporated into the structure of the DNAdendrimer has been demonstrated. For example, gold particles containingsurface immobilized streptavidin or anti-biotin antibody are used tobind to DNA dendrimers containing incorporated biotin moieties. Further,gold or other types of particles containing immobilized nucleic acidprobes have been used to bind to complementary nucleic acid sequences onDNA dendrimers. Additionally, other types of particles, including latexparticles, Q-Dots, carbon nanotubes, silver particles or silver coatedparticles, and others may be used for visible signal generation whencapable of binding to DNA dendrimers. DNA dendrimer binding particlesmay also be used for generating non-visible signals, for example Q-Dotsand latex particles containing fluorescent dyes for fluorescent assays.

In some embodiments, the DNA dendrimers are directly labeled withsignaling moieties (and signal generating moieties such as enzymes),thereby avoiding the use of signal generating particles, where suchdendrimer-label complexes are fully soluble in aqueous and othersolutions. There are clear advantages for using soluble DNA dendrimersin POC assays, including the reduction of steps achieved by avoiding theuse of an insoluble signal generating particles, the inherent stabilityof the soluble DNA dendrimer either in liquid form or in a dehydratedform as compared to particle conjugates, the ease and efficiency ofre-hydration of the DNA dendrimer after being dried to a POC assaysubstrate, and others.

In some embodiments, the DNA dendrimers used in POC assays aremanufactured in a manner which eliminates the need for covalentcrosslinking of label moieties and targeting devices to the DNAdendrimer as required for other previously disclosed DNA dendrimer assayapplications. Typically, in other assay formats including the use of DNAdendrimers on DNA and protein microarrays, DNA dendrimers have had labelmoieties and targeting devices covalently crosslinked and have beenultimately purified using a relatively inefficient sucrose gradientcentrifugation purification method previously disclosed (See e.g., U.S.Pat. No. 5,175,270 and U.S. Pat. No. 5,487,973). We have empiricallydetermined that DNA dendrimers which have not been crosslinked after theaddition of label moieties or targeting devices perform as well as DNAdendrimers prepared in the customary manufacturing process. This was asurprising finding which will significantly reduce the cost of producingsaid DNA dendrimers for use in a variety of POC lateral flow assayformats.

The present invention is further illustrated by the following exemplaryembodiments:

1. A test device for detecting an analyte in a liquid sample, whichdevice comprises a porous matrix that comprises a test zone on saidporous matrix, said test zone comprising a test reagent that binds to ananalyte or another binding reagent that binds to said analyte, or is ananalyte or an analyte analog that competes with an analyte in saidsample for binding to a binding reagent for said analyte,

wherein a liquid sample flows laterally along said test device andpasses (or a liquid sample is capable of flowing laterally along saidtest device and passing) said test zone to form a detectable signal toindicate presence, absence and/or amount of said analyte in said liquidsample, the formation of said detectable signal requires the use of adetectable label and a DNA dendrimer, said DNA dendrimer comprises afirst component that links said DNA dendrimer to a binding reagentlinkable to said DNA dendrimer, an analyte linkable to said DNAdendrimer, or an analyte analog linkable to said DNA dendrimer, and asecond component that links said DNA dendrimer to said detectable labellinkable to said DNA dendrimer, and wherein:

a) said analyte is not a polynucleotide; or

b) said DNA dendrimer comprises from about 1 to about 324, preferablyfrom about 60 to about 324, from about 80 to about 300 or from about 100to about 200, said first components; or

c) said DNA dendrimer, before said test device is used, is dried on alocation on said test device upstream from said test zone.

In some embodiments, said analyte is not a polynucleotide and said DNAdendrimer comprises from about 1 to about 324, preferably from about 60to about 324, from about 80 to about 300 or from about 100 to about 200,said first components. In other embodiments, said analyte is not apolynucleotide and said DNA dendrimer, before said test device is used,is dried on a location on said test device upstream from said test zone.In still other embodiments, said DNA dendrimer comprises from about 1 toabout 324, preferably from about 60 to about 324, from about 80 to about300 or from about 100 to about 200, said first components and said DNAdendrimer, before said test device is used, is dried on a location onsaid test device upstream from said test zone. In yet other embodiments,said analyte is not a polynucleotide, said DNA dendrimer comprises fromabout 1 to about 324, preferably from about 60 to about 324, from about80 to about 300 or from about 100 to about 200, said first componentsand said DNA dendrimer, before said test device is used, is dried on alocation on said test device upstream from said test zone.

2. The test device of embodiment 1, wherein the first component and thesecond component are the same.

3. The test device of embodiment 1, wherein the first component and thesecond component are different.

4. The test device of embodiment 3, wherein one of the first componentand the second component is a polynucleotide and the other is anon-polynucleotide moiety.

5. The test device of embodiment 4, wherein the first component is apolynucleotide and the second component is a non-polynucleotide moiety.

6. The test device of any of the embodiments 1-5, wherein thenon-polynucleotide moiety is a polypeptide.

7. The test device of any of the embodiments 1-6, wherein the firstcomponent links the DNA dendrimer to a binding reagent that binds to ananalyte.

8. The test device of any of the embodiments 1-6, wherein the firstcomponent links the DNA dendrimer to a binding reagent that binds toanother binding reagent that binds to an analyte.

9. The test device of any of the embodiments 1-8, wherein the secondcomponent links the DNA dendrimer to a detectable label.

10. The test device of any of the embodiments 1-8, wherein the secondcomponent links the DNA dendrimer to another binding reagent that islinked to a detectable label.

11. The test device of any of the embodiments 1-10, which is to be usedin a sandwich assay for the analyte and wherein the test reagent at thetest zone binds to the analyte, a second binding reagent that binds tothe analyte is used, the second binding reagent comprises apolynucleotide that is substantially complementary to a polynucleotidethat is the first component of a DNA dendrimer.

12. The test device of any of the embodiments 1-10, which is to be usedin a sandwich assay for the analyte and wherein the test reagent at thetest zone binds to the analyte, a second binding reagent that binds toanother binding reagent that binds to an analyte is used, the secondbinding reagent comprises a polynucleotide that is substantiallycomplementary to a polynucleotide that is the first component of a DNAdendrimer.

13. The test device of any of the embodiments 1-10, which is to be usedin a competitive assay for the analyte and wherein the test reagent atthe test zone is an analyte or an analyte analog, a second bindingreagent that binds to the analyte is used, the second binding reagentcomprises a polynucleotide that is substantially complementary to apolynucleotide that is the first component of a DNA dendrimer, and theanalyte or an analyte analog at the test zone competes with an analytein the sample for binding to the second binding reagent.

14. The test device of any of the embodiments 1-10, which is to be usedin a competitive assay for the analyte and wherein the test reagent atthe test zone is an analyte or an analyte analog, a second bindingreagent that binds to another binding reagent that binds to an analyteis used, the second binding reagent comprises a polynucleotide that issubstantially complementary to a polynucleotide that is the firstcomponent of a DNA dendrimer, and the analyte or an analyte analog atthe test zone competes with an analyte in the sample for binding to thebinding reagent that is bound to the second binding reagent.

15. The test device of any of the embodiments 1-14, wherein the analyte,analyte analog, test reagent and/or binding reagent is an inorganicmolecule, an organic molecule or a complex thereof.

16. The test device of embodiment 15, wherein the organic molecule isselected from the group consisting of an amino acid, a peptide, aprotein, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid,a vitamin, a monosaccharide, an oligosaccharide, a carbohydrate, a lipidand a complex thereof.

17. The test device of any of the embodiments 1-14, wherein the analyteis a polypeptide or a small molecule, and the test reagent and/orbinding reagent that binds to the analyte is an antibody that binds tothe polypeptide or small molecule.

18. The test device of any of the embodiments 1-14, wherein the analyteis a polynucleotide, and the test reagent and/or binding reagent thatbinds to the analyte is another polynucleotide that is substantiallycomplementary to the analyte polynucleotide.

19. The test device of any of the embodiments 1-18, wherein the matrixcomprises nitrocellulose, glass fiber, polypropylene, polyethylene(preferably of very high molecular weight), polyvinylidene flouride,ethylene vinylacetate, acrylonitrile and/or polytetrafluoro-ethylene.

20. The test device of any of the embodiments 1-19, wherein the matrixis in the form a strip or a circle.

21. The test device of any of the embodiments 1-20, wherein the matrixis a single element or comprises multiple elements.

22. The test device of any of the embodiments 1-21, which furthercomprises a sample application element upstream from and in fluidcommunication with the matrix.

23. The test device of any of the embodiments 1-22, which furthercomprises a liquid absorption element downstream from and in fluidcommunication with the matrix.

24. The test device of any of the embodiments 1-23, which furthercomprises a control zone comprising means for indicating proper flow ofthe liquid sample and/or a valid test result.

25. The test device of any of the embodiments 1-24, wherein at least aportion of the matrix is supported by a solid backing.

26. The test device of any of the embodiments 1-25, wherein a substanceis dried on a portion of the matrix upstream from the test zone, thedried substance being capable of being moved by a liquid sample and/or afurther liquid to the test zone and/or a control zone to generate adetectable signal, the dried substance being at least one of:

1) the DNA dendrimer;

2) the first binding reagent that binds to the analyte, the secondbinding reagent that binds to another binding reagent that binds to theanalyte, the analyte or the analyte analog, each of the first bindingreagent, second binding reagent, analyte or analyte analog beinglinkable to the DNA dendrimer; and

-   -   3) the detectable label linkable to the DNA dendrimer.

27. The test device of embodiment 26, wherein two substances are driedon a portion of the matrix upstream from the test zone, the driedsubstances being capable of being moved by a liquid sample and/or afurther liquid to the test zone and/or a control zone to generate adetectable signal, the dried substances being at least two of:

1) the DNA dendrimer;

2) the first binding reagent that binds to the analyte, the secondbinding reagent that binds to another binding reagent that binds to theanalyte, the analyte or the analyte analog, each of the first bindingreagent, second binding reagent, analyte or analyte analog beinglinkable to the DNA dendrimer; and

3) the label linkable to the DNA dendrimer;

are dried on a portion of the matrix upstream from the test zone, thedried substances, whether separately or in complex, being capable ofbeing moved by a liquid sample and/or a further liquid to the test zoneand/or a control zone to generate a detectable signal.

28. The test device of embodiment 26, wherein three substances are driedon a portion of the matrix upstream from the test zone, the driedsubstances being capable of being moved by a liquid sample and/or afurther liquid to the test zone and/or a control zone to generate adetectable signal, the dried substances being all of:

1) the DNA dendrimer;

2) the first binding reagent that binds to the analyte, the secondbinding reagent that binds to another binding reagent that binds to theanalyte, the analyte or the analyte analog, each of the first bindingreagent, second binding reagent, analyte or analyte analog beinglinkable to the DNA dendrimer; and

3) the label linkable to the DNA dendrimer.

29. The test device of any of the embodiments 26-28, wherein thesubstance(s) is dried on a conjugate element that is upstream from thetest zone.

30. The test device of any of the embodiments 26-29, wherein thesubstance(s) is located downstream from a sample application place onthe test device.

31. The test device of any of the embodiments 26-30, wherein thesubstance(s) is located upstream from a sample application place on thetest device.

32. The test device of any of the embodiments 1-31, wherein thedetectable label is a soluble label.

33. The test device of embodiments 32, wherein the soluble label is asoluble enzyme or fluorescent label.

34. The test device of any of the embodiments 1-31, wherein thedetectable label is a particle label.

35. The test device of embodiments 34, wherein the particle label is avisible or a non-visible particle label.

36. The test device of embodiments 35, wherein the visible particlelabel is selected from the group consisting of a gold particle, a latexparticle, a Q-Dot, a carbon nanotube, a silver particle, a silver coatedparticle and a complex thereof.

37. The test device of embodiments 35, wherein the non-visible particlelabel is a fluorescent particle.

38. The test device of any of the embodiments 26-37, wherein thesubstance(s) is dried in the presence of a material that: a) stabilizesthe dried substance(s); b) facilitates solubilization or resuspension ofthe dried substance(s) in a liquid; and/or c) facilitates mobility ofthe dried substance(s).

39. The test device of embodiments 38, wherein the material is selectedfrom the group consisting of a protein, a peptide, a polysaccharide, asugar, a polymer, a gelatin and a detergent.

40. The test device of any of the embodiments 1-39, wherein a sampleliquid alone is used to transport the analyte and/or the substance(s) tothe test zone.

41. The test device of any of the embodiments 1-39, wherein a developingliquid is used to transport the analyte and/or the substance(s) to thetest zone.

42. The test device of any of the embodiments 1-41, which furthercomprises a housing that covers at least a portion of the test device,wherein the housing comprises a sample application port to allow sampleapplication upstream from or to the test zone and an optic openingaround the test zone to allow signal detection at the test zone.

43. The test device of embodiment 42, wherein the housing covers theentire test device.

44. The test device of embodiment 42, wherein at least a portion of thesample receiving portion of the matrix or the sample application elementis not covered by the housing and a sample is applied to the portion ofthe sample receiving portion of the matrix or the sample applicationelement outside the housing and then transported to the test zone.

45. The test device of any of the embodiments 1-44, wherein none of:

1) the DNA dendrimer;

2) the first binding reagent that binds to the analyte, the secondbinding reagent that binds to another binding reagent that binds to theanalyte, the analyte or the analyte analog, each of the first bindingreagent, second binding reagent, analyte or analyte analog beinglinkable to the DNA dendrimer; and

3) the detectable label linkable to the DNA dendrimer;

is covalently bound or crosslinked to each other.

46. The test device of any of the embodiments 1-44, wherein at least twoof:

1) the DNA dendrimer;

2) the first binding reagent that binds to the analyte, the secondbinding reagent that binds to another binding reagent that binds to theanalyte, the analyte or the analyte analog, each of the first bindingreagent, second binding reagent, analyte or analyte analog beinglinkable to the DNA dendrimer; and

3) the detectable label linkable to the DNA dendrimer;

are covalently bound or crosslinked to each other.

47. The test device of any of the embodiments 1-44, wherein all of:

1) the DNA dendrimer;

2) the first binding reagent that binds to the analyte, the secondbinding reagent that binds to another binding reagent that binds to theanalyte, the analyte or the analyte analog, each of the first bindingreagent, second binding reagent, analyte or analyte analog beinglinkable to the DNA dendrimer; and

3) the detectable label linkable to the DNA dendrimer;

are covalently bound or crosslinked to each other.

48. The test device of any of the embodiments 1-47, wherein the DNAdendrimer comprises from about 10 to about 1,500, preferably from about40 to about 1,500, from about 900 to about 1,100, second components thatlink the DNA dendrimer to a detectable label linkable to the DNAdendrimer.

49. The test device of any of the embodiments 1-48, wherein the DNAdendrimer comprises from about 400 to about 80,000, preferably fromabout 4,000 to about 80,000, from about 60,000 to about 80,000, DNAnucleotides.

50. The test device of any of the embodiments 1-49, wherein the DNAdendrimer comprises a one-layer, a two-layer, a three-layer or afour-layer structure, or a DNA dendrimer structure comprising at leastone monomeric unit as described in U.S. Pat. No. 6,274,723B1.

51. The test device of any of the embodiments 1-50, wherein the liquidsample has moved laterally along the test device to generate adetectable signal at the test zone.

52. A method for detecting an analyte in a liquid sample, which methodcomprises:

a) contacting a liquid sample with the test device of any of theembodiments 1-50, wherein the liquid sample is applied to a site of thetest device upstream of the test zone;

b) transporting an analyte, if present in the liquid sample, adetectable label and a DNA dendrimer to the test zone; and

c) assessing the presence, absence, and/or amount of a signal generatedby the detectable label at the test zone to determining the presence,absence and/or amount of the analyte in the liquid sample.

53. The method of embodiment 52, wherein the liquid sample and at leastone of:

1) the DNA dendrimer;

2) the first binding reagent that binds to the analyte, the secondbinding reagent that binds to another binding reagent that binds to theanalyte, the analyte or the analyte analog, each of the first bindingreagent, second binding reagent, analyte or analyte analog beinglinkable to the DNA dendrimer; and

3) the detectable label linkable to the DNA dendrimer;

are premixed to form a mixture and the mixture is applied to the testdevice.

54. The method of embodiment 52, wherein the liquid sample and at leasttwo of:

1) the DNA dendrimer;

2) the first binding reagent that binds to the analyte, the secondbinding reagent that binds to another binding reagent that binds to theanalyte, the analyte or the analyte analog, each of the first bindingreagent, second binding reagent, analyte or analyte analog beinglinkable to the DNA dendrimer; and

3) the detectable label linkable to the DNA dendrimer;

are premixed to form a mixture and the mixture is applied to the testdevice.

55. The method of embodiment 52, wherein the liquid sample and all of:

1) the DNA dendrimer;

2) the first binding reagent that binds to the analyte, the secondbinding reagent that binds to another binding reagent that binds to theanalyte, the analyte or the analyte analog, each of the first bindingreagent, second binding reagent, analyte or analyte analog beinglinkable to the DNA dendrimer; and

3) the detectable label linkable to the DNA dendrimer;

are premixed to form a mixture and the mixture is applied to the testdevice.

56. The method of any of the embodiments 52-55, which is conducted in aliquid comprising from about 0.001% (v/v) to about 5% (v/v), preferably,from about 0.01% (v/v) to about 0.5% (v/v), or at about 0.01% (v/v) orless Tween-20.

57. The method of any of the embodiments 52-56, which is conducted in aliquid comprising dextran sulfate.

58. The method of embodiment 52, wherein a substance is dried on aportion of the test device upstream from the test zone, the driedsubstance is solubilized or resuspended, and transported to the testzone and/or a control zone to generate a detectable signal, the driedsubstance being at least one of:

1) the DNA dendrimer;

2) the first binding reagent that binds to the analyte, the secondbinding reagent that binds to another binding reagent that binds to theanalyte, the analyte or the analyte analog, each of the first bindingreagent, second binding reagent, analyte or analyte analog beinglinkable to the DNA dendrimer; and

3) the detectable label linkable to the DNA dendrimer.

59. The method of embodiment 58, wherein the dried substance is locateddownstream from the sample application site, and the dried substance issolubilized or resuspended, and transported to the test zone and/or acontrol zone by the liquid sample.

60. The method of embodiment 58, wherein the dried substance is locatedupstream from the sample application site, and the dried substance issolubilized or resuspended, and transported to the test zone and/or acontrol zone by another liquid.

61. The method of embodiment 58, wherein the dried substance issolubilized or resuspended, and transported to the test zone and/or acontrol zone by the liquid sample alone.

62. The method of embodiment 58, wherein the analyte and/or driedsubstance is solubilized or resuspended, and transported to the testzone and/or a control zone by another liquid.

63. The method of any of the embodiments 52-62, wherein the liquidsample is a body fluid sample.

64. The method of embodiment 63, wherein the body fluid sample isselected from the group consisting of a whole blood, a serum, a plasmaand a urine sample.

65. The method of any of the embodiments 52-64, wherein the liquidsample is a sample derived from a biological, a forensics, a food, abiowarfare, or an environmental source.

66. The method of any of the embodiments 52-65, which is used toquantify or semi-quantify the amount of an analyte in a liquid sample.

67. The method of any of the embodiments 52-66, which is used to detectmultiple analytes in a liquid sample.

68. The method of embodiment 67, which is used to quantify orsemi-quantify the amounts of the multiple analytes in the liquid sample.

69. The method of any of the embodiments 52-68, wherein the analyte isselected from the group consisting of a cell, a virus and a molecule.

70. A test device for detecting an analyte in a liquid sample, whichdevice comprises a porous matrix that comprises a test zone on saidporous matrix, said test zone comprising a test reagent that binds to ananalyte or another binding reagent that binds to said analyte, or is ananalyte or an analyte analog that competes with an analyte in saidsample for binding to a binding reagent for said analyte,

wherein a liquid sample flows laterally along said test device andpasses (or a liquid sample is capable of flowing laterally along saidtest device and passing) said test zone to form a detectable signal toindicate presence, absence and/or amount of said analyte in said liquidsample, the formation of said detectable signal requires the use of aDNA dendrimer linked to a detectable label covalently or non-covalently,and said DNA dendrimer comprises a component that links said DNAdendrimer to a binding reagent linkable to said DNA dendrimer, ananalyte linkable to said DNA dendrimer, or an analyte analog linkable tosaid DNA dendrimer, and wherein:

a) said analyte is not a polynucleotide; or

b) said DNA dendrimer comprises from about 1 to about 324, preferablyfrom about 60 to about 324, from about 80 to about 300 or from about 100to about 200, said first components; or

c) said DNA dendrimer, before said test device is used, is dried on alocation on said test device upstream from said test zone.

In some embodiments, said analyte is not a polynucleotide and said DNAdendrimer comprises from about 1 to about 324, preferably from about 60to about 324, from about 80 to about 300 or from about 100 to about 200,said first components. In other embodiments, said analyte is not apolynucleotide and said DNA dendrimer, before said test device is used,is dried on a location on said test device upstream from said test zone.In still other embodiments, said DNA dendrimer comprises from about 1 toabout 324, preferably from about 60 to about 324, from about 80 to about300 or from about 100 to about 200, said first components and said DNAdendrimer, before said test device is used, is dried on a location onsaid test device upstream from said test zone. In yet other embodiments,said analyte is not a polynucleotide, said DNA dendrimer comprises fromabout 1 to about 324, preferably from about 60 to about 324, from about80 to about 300 or from about 100 to about 200, said first componentsand said DNA dendrimer, before said test device is used, is dried on alocation on said test device upstream from said test zone.

71. The test device of embodiment 70, wherein the component is apolynucleotide.

72. The test device of embodiment 70 or 71, which is to be used in asandwich assay for the analyte and wherein the test reagent at the testzone binds to the analyte, a second binding reagent that binds to theanalyte is used, the second binding reagent comprises a polynucleotidethat is substantially complementary to a polynucleotide that is thecomponent of a DNA dendrimer. 73. The test device of embodiment 70 or71, which is to be used in a sandwich assay for the analyte and whereinthe test reagent at the test zone binds to the analyte, a second bindingreagent that binds to another binding reagent that binds to an analyteis used, the second binding reagent comprises a polynucleotide that issubstantially complementary to a polynucleotide that is the component ofa DNA dendrimer.

74. The test device of embodiment 70 or 71, which is to be used in acompetitive assay for the analyte and wherein the test reagent at thetest zone is an analyte or an analyte analog, a second binding reagentthat binds to the analyte is used, the second binding reagent comprisesa polynucleotide that is substantially complementary to a polynucleotidethat is the component of a DNA dendrimer, and the analyte or an analyteanalog at the test zone competes with an analyte in the sample forbinding to the second binding reagent.

75. The test device of embodiment 70 or 71, which is to be used in acompetitive assay for the analyte and wherein the test reagent at thetest zone is an analyte or an analyte analog, a second binding reagentthat binds to another binding reagent that binds to an analyte is used,the second binding reagent comprises a polynucleotide that issubstantially complementary to a polynucleotide that is the component ofa DNA dendrimer, and the analyte or an analyte analog at the test zonecompetes with an analyte in the sample for binding to the bindingreagent that is bound to the second binding reagent.

76. The test device of any of the embodiments 70-75, wherein theanalyte, analyte analog, test reagent and/or binding reagent is aninorganic molecule, an organic molecule or a complex thereof.

77. The test device of embodiment 76, wherein the organic molecule isselected from the group consisting of an amino acid, a peptide, aprotein, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid,a vitamin, a monosaccharide, an oligosaccharide, a carbohydrate, a lipidand a complex thereof.

78. The test device of any of the embodiments 70-77, wherein the analyteis a polypeptide or a small molecule, and the test reagent and/orbinding reagent that binds to the analyte is an antibody that binds tothe polypeptide or small molecule.

79. The test device of any of the embodiments 70-77, wherein the analyteis a polynucleotide, and the test reagent and/or binding reagent thatbinds to the analyte is another polynucleotide that is substantiallycomplementary to the analyte polynucleotide.

80. The test device of any of the embodiments 70-79, wherein the matrixcomprises nitrocellulose, glass fiber, polypropylene, polyethylene(preferably of very high molecular weight), polyvinylidene flouride,ethylene vinylacetate, acrylonitrile and/or polytetrafluoro-ethylene.

81. The test device of any of the embodiments 70-80, wherein the matrixis in the form a strip or a circle.

82. The test device of any of the embodiments 70-81, wherein the matrixis a single element or comprises multiple elements.

83. The test device of any of the embodiments 70-82, which furthercomprises a sample application element upstream from and in fluidcommunication with the matrix.

84. The test device of any of the embodiments 70-83, which furthercomprises a liquid absorption element downstream from and in fluidcommunication with the matrix.

85. The test device of any of the embodiments 70-84, which furthercomprises a control zone comprising means for indicating proper flow ofthe liquid sample and/or a valid test result.

86. The test device of any of the embodiments 70-85, wherein at least aportion of the matrix is supported by a solid backing.

87. The test device of any of the embodiments 70-86, wherein a substanceis dried on a portion of the matrix upstream from the test zone, thedried substance being capable of being moved by a liquid sample and/or afurther liquid to the test zone and/or a control zone to generate adetectable signal, the dried substance being at least one of:

1) the DNA dendrimer; and

2) the first binding reagent that binds to the analyte, the secondbinding reagent that binds to another binding reagent that binds to theanalyte, the analyte or the analyte analog, each of the first bindingreagent, second binding reagent, analyte or analyte analog beinglinkable to the DNA dendrimer.

88. The test device of embodiments 87, wherein both of the DNAdendrimer, and the first binding reagent that binds to the analyte, thesecond binding reagent that binds to another binding reagent that bindsto the analyte, the analyte or the analyte analog, are dried on aportion of the matrix upstream from the test zone.

89. The test device of embodiment 87 or 88, wherein the substance(s) isdried on a conjugate element that is upstream from the test zone.

90. The test device of embodiment 87 or 88, wherein the substance(s) islocated downstream from a sample application place on the test device.

91. The test device of embodiment 87 or 88, wherein the substance(s) islocated upstream from a sample application place on the test device.

92. The test device of any of the embodiments 70-91, wherein thedetectable label is a soluble label.

93. The test device of embodiments 92, wherein the soluble label is asoluble enzyme or fluorescent label.

94. The test device of any of the embodiments 70-91, wherein thedetectable label is a particle label.

95. The test device of embodiments 94, wherein the particle label is avisible or a non-visible particle label.

96. The test device of embodiments 95, wherein the visible particlelabel is selected from the group consisting of a gold particle, a latexparticle, a Q-Dot, a carbon nanotube, a silver particle, a silver coatedparticle and a complex thereof.

97. The test device of embodiments 95, wherein the non-visible particlelabel is a fluorescent particle.

98. The test device of any of the embodiments 87-97, wherein thesubstance(s) is dried in the presence of a material that: a) stabilizesthe dried substance(s); b) facilitates solubilization or resuspension ofthe dried substance(s) in a liquid; and/or c) facilitates mobility ofthe dried substance(s).

99. The test device of embodiments 98, wherein the material is selectedfrom the group consisting of a protein, a peptide, a polysaccharide, asugar, a polymer, a gelatin and a detergent.

100. The test device of any of the embodiments 70-99, wherein a sampleliquid alone is used to transport the analyte and/or the substance(s) tothe test zone.

101. The test device of any of the embodiments 70-99, wherein adeveloping liquid is used to transport the analyte and/or thesubstance(s) to the test zone.

102. The test device of any of the embodiments 70-101, which furthercomprises a housing that covers at least a portion of the test device,wherein the housing comprises a sample application port to allow sampleapplication upstream from or to the test zone and an optic openingaround the test zone to allow signal detection at the test zone.

103. The test device of embodiment 102, wherein the housing covers theentire test device.

104. The test device of embodiment 102, wherein at least a portion ofthe sample receiving portion of the matrix or the sample applicationelement is not covered by the housing and a sample is applied to theportion of the sample receiving portion of the matrix or the sampleapplication element outside the housing and then transported to the testzone.

105. The test device of any of the embodiments 70-104, wherein the DNAdendrimer comprises from about 1 to about 324, preferably from about 60to about 324, from about 80 to about 300 or from about 100 to about 200,said first components.

106. The test device of any of the embodiments 70-105, wherein the DNAdendrimer comprises from about 10 to about 1,500, preferably from about40 to about 1,500 or from about 900 to about 1,100, the detectablelabel.

107. The test device of any of the embodiments 70-106, wherein the DNAdendrimer comprises from about 400 to about 80,000, preferably fromabout 4,000 to about 80,000, or from about 60,000 to about 80,000, DNAnucleotides.

108. The test device of any of the embodiments 70-107, wherein the DNAdendrimer comprises a one-layer, a two-layer, a three-layer or afour-layer structure, or a DNA dendrimer structure comprising at leastone monomeric unit as described in U.S. Pat. No. 6,274,723B1.

109. The test device of any of the embodiments 70-108, wherein theliquid sample has moved laterally along the test device to generate adetectable signal at the test zone.

110. A method for detecting an analyte in a liquid sample, which methodcomprises:

a) contacting a liquid sample with the test device of any of theembodiments 70-108, wherein the liquid sample is applied to a site ofthe test device upstream of the test zone;

b) transporting an analyte, if present in the liquid sample, adetectable label and a DNA dendrimer to the test zone; and

c) assessing the presence, absence, and/or amount of a signal generatedby the detectable label at the test zone to determining the presence,absence and/or amount of the analyte in the liquid sample.

111. The method of embodiment 110, wherein the liquid sample and atleast one of:

1) the DNA dendrimer linked to a detectable label non-covalently; and

2) the first binding reagent that binds to the analyte, the secondbinding reagent that binds to another binding reagent that binds to theanalyte, the analyte or the analyte analog, each of the first bindingreagent, second binding reagent, analyte or analyte analog beinglinkable to the DNA dendrimer;

are premixed to form a mixture and the mixture is applied to the testdevice.

112. The method of embodiment 110, wherein the liquid sample and bothof:

1) the DNA dendrimer linked to a detectable label non-covalently; and

2) the first binding reagent that binds to the analyte, the secondbinding reagent that binds to another binding reagent that binds to theanalyte, the analyte or the analyte analog, each of the first bindingreagent, second binding reagent, analyte or analyte analog beinglinkable to the DNA dendrimer;

are premixed to form a mixture and the mixture is applied to the testdevice.

113. The method of any of the embodiments 110-112, which is conducted ina liquid comprising from about 0.001% (v/v) to about 5% (v/v),preferably, from about 0.01% (v/v) to about 0.5% (v/v), or at about0.01% (v/v) or less Tween-20.

114. The method of any of the embodiments 110-113, which is conducted ina liquid comprising dextran sulfate.

115. The method of embodiment 110, wherein a substance is dried on aportion of the test device upstream from the test zone, the driedsubstance is capable of being moved by a liquid sample and/or a furtherliquid to the test zone and/or a control zone to generate a detectablesignal, the dried substance being at least one of:

1) the DNA dendrimer linked to a detectable label non-covalently; and

2) the first binding reagent that binds to the analyte, the secondbinding reagent that binds to another binding reagent that binds to theanalyte, the analyte or the analyte analog, each of the first bindingreagent, second binding reagent, analyte or analyte analog beinglinkable to the DNA dendrimer.

116. The method of embodiment 115, wherein the dried substance islocated downstream from the sample application site, and the driedsubstance is solubilized or resuspended, and transported to the testzone and/or a control zone by the liquid sample.

117. The method of embodiment 115, wherein the dried substance islocated upstream from the sample application site, and the driedsubstance is solubilized or resuspended, and transported to the testzone and/or a control zone by another liquid.

118. The method of embodiment 115, wherein the dried substance issolubilized or resuspended, and transported to the test zone and/or acontrol zone by the liquid sample alone.

119. The method of embodiment 115, wherein the analyte and/or driedsubstance is solubilized or resuspended, and transported to the testzone and/or a control zone by another liquid.

120. The method of any of the embodiments 110-119, wherein the liquidsample is a body fluid sample.

121. The method of embodiment 120, wherein the body fluid sample isselected from the group consisting of a whole blood, a serum, a plasmaand a urine sample.

122. The method of any of the embodiments 110-121, wherein the liquidsample is a sample derived from a biological, a forensics, a food, abiowarfare, or an environmental source.

123. The method of any of the embodiments 110-122, which is used toquantify or semi-quantify the amount of an analyte in a liquid sample.

124. The method of any of the embodiments 110-123, which is used todetect multiple analytes in a liquid sample.

125. The method of embodiment 124, which is used to quantify orsemi-quantify the amounts of the multiple analytes in the liquid sample.

126. The method of any of the embodiments 110-125, wherein the analyteis selected from the group consisting of a cell, a virus and a molecule.

127. The test device of any of embodiments 1-51 and 70-109, wherein theDNA dendrimer, before the test device is used, is dried on a location onthe test device upstream from the test zone, and at least about 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%of the dried DNA dendrimer is capable of being moved to the test zone bya liquid.

128. The test device of embodiment 127, wherein the DNA dendrimer isdried on a location on the test device in the presence of a materialthat: a) stabilizes the dried DNA dendrimer; b) facilitatessolubilization or resuspension of the dried DNA dendrimer in a liquid;and/or c) facilitates mobility of the dried DNA dendrimer.

129. The test device of embodiment 128, wherein the material is selectedfrom the group consisting of a protein, a peptide, a polysaccharide, asugar, a polymer, a gelatin and a detergent.

130. The test device of embodiment 129, wherein the sugar is trehaloseand/or sucrose.

131. The test device of any of embodiments 127-130, wherein the liquidcomprises a material that: a) stabilizes the dried DNA dendrimer; b)facilitates solubilization or resuspension of the dried DNA dendrimer inthe liquid; and/or c) facilitates mobility of the dried DNA dendrimer.

132. The test device of embodiment 131, wherein the material is selectedfrom the group consisting of a protein, a peptide, a polysaccharide, asugar, a polymer, a gelatin and a detergent.

133. The test device of embodiment 132, wherein the protein is BSA andthe detergent is Tween-20.

134. The test device of any of embodiments 127-133, wherein the liquidhas a volume of at least about 20 ul, 25 ul, 30 ul, 35 ul, 40 ul, 45 ul,50 ul, 55 ul, 60 ul, 65 ul, 70 ul, 75 ul, 80 ul, 85 ul, 90 ul, 95 ul,100 ul, 110 ul, 120 ul, 150 ul, 200 ul, 300 ul, 400 ul, 500 ul, or avolume larger than 500 ul.

135. The test device of any of embodiments 1-51, 70-109 and 127-134,wherein the DNA dendrimer comprises:

a) an one-layer DNA dendrimer that comprises from about 1-2, 2-4 or 4-8of the first and/or second component(s);

b) a two-layer DNA dendrimer that comprises from about 1-4, 4-8 or 8-18of the first and/or second component(s);

c) a three-layer DNA dendrimer that comprises from about 2-6, 6-10,10-18, 18-28, 28-38, 38-50 or more than 50 of the first and/or secondcomponents; and/or

d) a four-layer DNA dendrimer that comprises from about 15-25, 25-45,45-60, 60-80, 85-105, 105-130 or more than 130 of the first and/orsecond components.

136. The test device of embodiment 135, wherein at least one of thefirst component(s) and the second component(s) is a polynucleotide andthe other is a non-polynucleotide moiety.

137. The test device of embodiment 135, wherein the DNA dendrimercomprises:

a) an one-layer DNA dendrimer that comprises from about 1-2, 2-4 or 4-8of the first component(s);

b) a two-layer DNA dendrimer that comprises from about 1-4, 4-8 or 8-18of the first component(s);

c) a three-layer DNA dendrimer that comprises from about 2-6, 6-10,10-18, 18-28, 28-38, 38-50 or more than 50 of the first components;and/or

d) a four-layer DNA dendrimer that comprises from about 15-25, 25-45,45-60, 60-80, 85-105, 105-130 or more than 130 of the first components.

138. The test device of embodiment 137, wherein the first component(s)comprise polynucleotide(s).

139. The test device of embodiment 138, wherein the polynucleotide(s)are DNA strand(s).

140. A method for detecting an analyte in a liquid sample, which methodcomprises:

a) contacting a liquid sample with the test device of any of embodiments127-139, wherein the liquid sample is applied to a site of the testdevice upstream of the test zone;

b) transporting an analyte, if present in the liquid sample, adetectable label and a DNA dendrimer to the test zone; and

c) assessing the presence, absence, and/or amount of a signal generatedby the detectable label at the test zone to determining the presence,absence and/or amount of the analyte in the liquid sample.

141. The test device of embodiment 140, wherein the signal generated bythe detectable label at the test zone is enhanced by:

a) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 foldsor higher relative to a signal generated by the detectable label at thetest zone using a DNA dendrimer comprising a smaller number of the firstand/or second component(s); or

b) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30,40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200 folds or higher relative to a signal generated by thedetectable label at the test zone without using a DNA dendrimer.

142. A test device for detecting an analyte in a liquid sample, whichdevice comprises a porous matrix that comprises a test zone on saidporous matrix, said test zone comprising a test reagent that binds to ananalyte or another binding reagent that binds to said analyte, or is ananalyte or an analyte analog that competes with an analyte in saidsample for binding to a binding reagent for said analyte,

wherein a liquid sample is capable of flowing laterally along said testdevice and passing said test zone to form a detectable signal toindicate presence, absence and/or amount of said analyte in said liquidsample,

wherein:

a) the formation of said detectable signal requires the use of adetectable label and a DNA dendrimer, said DNA dendrimer comprises afirst component that links said DNA dendrimer to a binding reagentlinkable to said DNA dendrimer, an analyte linkable to said DNAdendrimer, or an analyte analog linkable to said DNA dendrimer, and asecond component that links said DNA dendrimer to said detectable labellinkable to said DNA dendrimer, or

b) the formation of said detectable signal requires the use of a DNAdendrimer linked to a detectable label covalently or non-covalently, andsaid DNA dendrimer comprises a third component that links said DNAdendrimer to a binding reagent linkable to said DNA dendrimer, ananalyte linkable to said DNA dendrimer, or an analyte analog linkable tosaid DNA dendrimer, and wherein:

c) said analyte is not a polynucleotide; and/or

d) said DNA dendrimer comprises from about 1 to about 324, preferablyfrom about 60 to about 324, from about 80 to about 300 or from about 100to about 200, said first components; and/or

e) said DNA dendrimer, before said test device is used, is dried on alocation on said test device upstream from said test zone.

143. A method for detecting an analyte in a liquid sample, which methodcomprises:

a) contacting a liquid sample with the test device of embodiment 142,wherein the liquid sample is applied to a site of the test deviceupstream of the test zone;

b) transporting an analyte, if present in the liquid sample, adetectable label and a DNA dendrimer to the test zone; and

c) assessing the presence, absence, and/or amount of a signal generatedby the detectable label at the test zone to determining the presence,absence and/or amount of the analyte in the liquid sample.

144. A kit for detecting an analyte in a liquid sample, which kitcomprises:

a) a porous matrix that comprises a test zone on said porous matrix,said test zone comprising a test reagent that binds to an analyte oranother binding reagent that binds to said analyte, or is an analyte oran analyte analog that competes with an analyte in said sample forbinding to a binding reagent for said analyte; and

b) a DNA dendrimer, wherein:

-   -   1) said DNA dendrimer comprises a first component that links        said DNA dendrimer to a binding reagent linkable to said DNA        dendrimer, an analyte linkable to said DNA dendrimer, or an        analyte analog linkable to said DNA dendrimer, and a second        component that links said DNA dendrimer to said detectable label        linkable to said DNA dendrimer, or    -   2) said DNA dendrimer is linked to a detectable label covalently        or non-covalently and comprises a third component that links        said DNA dendrimer to a binding reagent linkable to said DNA        dendrimer, an analyte linkable to said DNA dendrimer, or an        analyte analog linkable to said DNA dendrimer.

145. The test device of embodiment 144, wherein the DNA dendrimercomprises from about 1 to about 324, preferably from about 60 to about324, from about 80 to about 300 or from about 100 to about 200, of thefirst or third components.

146. The kit of embodiment 144 or 145, wherein one of the firstcomponent and the second component is a polynucleotide and the other isa non-polynucleotide moiety.

147. The kit of embodiment 144 or 145, wherein the third component is apolynucleotide.

148. The kit of any of embodiments 144-147, wherein at least two of:

1) the DNA dendrimer;

2) the first binding reagent that binds to the analyte, the secondbinding reagent that binds to another binding reagent that binds to theanalyte, the analyte or the analyte analog, each of the first bindingreagent, second binding reagent, analyte or analyte analog beinglinkable to the DNA dendrimer; and

3) the detectable label linkable to the DNA dendrimer;

are covalently bound or crosslinked to each other.

149. The kit of any of embodiments 144-148, wherein the DNA dendrimercomprises from about 10 to about 1,500, preferably from about 40 toabout 1,500 or from about 900 to about 1,100, the detectable label.

150. The kit of any of embodiments 144-149, wherein the DNA dendrimercomprises from about 400 to about 80,000, preferably from about 4,000 toabout 80,000, or from about 60,000 to about 80,000, DNA nucleotides.

151. The kit of any of embodiments 144-150, wherein the DNA dendrimercomprises a one-layer, a two-layer, a three-layer or a four-layerstructure, or a DNA dendrimer structure comprising at least onemonomeric unit as described in U.S. Pat. No. 6,274,723B1.

152. The kit of any of embodiments 144-151, which further comprises oneor more of an antibody-oligo conjugate, an analyte capture antibody,streptavidin-label, e.g., gold particle, conjugate or other label,buffers, reagents, instructions etc.

G. EXAMPLES

The following examples represent applications of the above descriptionsof using DNA dendrimers in POC lateral flow assays. The followingexamples are included for illustrative purposes only and are notintended to limit the scope of the invention.

Example 1 Use of a DNA Dendrimer and an Anti-hCG Mouse MonoclonalTargeting Antibody-Oligo Conjugate to Improve Sensitivity in an allLiquid (“Wet”), Direct Sandwich POC Lateral Flow Assay

Manufacture of the DNA Dendrimer

DNA dendrimers are manufactured as previously disclosed (see U.S. Pat.Nos. 5,175,270, 5,484,904, 5,487,973, 6,110,687, and 6,274,723).Briefly, a DNA dendrimer is constructed from DNA monomers, each of whichis made from two DNA strands that share a region of sequencecomplementarity located in the central portion of each strand. When thetwo strands anneal to form the monomer the resulting structure can bedescribed as having a central double-stranded “waist” bordered by foursingle-stranded “arms”. This waist-plus-arms structure comprises thebasic DNA monomer. The single-stranded arms at the ends of each of thefive monomer types are designed to interact with one another in preciseand specific ways. Base-pairing between the arms of complementarymonomers allows directed assembly of the dendrimer through sequentialaddition of monomer layers (FIG. 3). Assembly of each layer of thedendrimer includes a cross-linking process where the strands of DNA arecovalently bonded to each other, thereby forming a completely covalentmolecule impervious to denaturing conditions that otherwise would causedeformation of the dendrimer structure (FIG. 2). In addition, 38 baseoligonucleotides that serve as complementary capture oligos arepre-ligated to the 5′ ends of an separate 124 mer single-stranded DNAoligonucleotide via a simple T4 DNA ligase-dependent ligation reaction(“strand ligate”), as follows:

124 mer DNA strand 500 nge(+)LIG-BR7 Bridging oligo (14 mer) 241.9 ngligA Cap03 oligo (38 mer) 306.5 ng10× Ligase buffer 1 uL (1/10 volume)Nuclease free water to 10 uL total volumeT4 DNA Ligase (1 U/uL) 1 uL (1 unit, 1/10 volume)

The first five reactants are added together, incubated at 42° C. for 15minutes and then allowed to cool to room temperature. The 6^(th)reactant is then added and incubated for 2 hours. The ligation reactionis stopped by adding 0.25 uL of 0.5M EDTA solution (to 12.5 mM final).This strand ligate is used in the manufacture of the capture sequencespecific biotinylated DNA dendrimer below.

The strand ligate (above) and DNA oligonucleotides containing biotinlabel moieties are covalently bound to the peripheral single strandedDNA sequences of the dendrimer via hybridization followed byintercalation of psoralen between the two hybridized strands of DNA. Theintercalated psoralen becomes covalently bound between the hybridizedDNA strands when exposed to 300 nm UV light.

A typical manufacturing hybridization-crosslinking process would be:

Add to a microfuge tube the following components:

4 layer DNA dendrimer 1000 ng (1 ug) strand ligate (from above) 500 ngas strand (total synthesis 10 uL) c(+) 2x biotin oligo (35mer) 452.4 ng(complementary to dendrimer “c arm” N3(−) #1 2x biotin oligo (18mer)291.9 ng (complementary to “strand 3 ligate”) N3(−) #2 2x biotin oligo(19mer) 243.2 ng (complementary to “strand 3 ligate”) N3(−) #3 2x biotinoligo (26mer) 316.2 ng (complementary to “strand 3 ligate”) 5M NaCl 1.1uL (0.2M Final) 0.1M DTT  0.7 uL (2.5 mM Final) Nuclease free Water to28 uL total volume 2,4,8 trimethyl psoralen saturated 1.1 uL solution inethanol

The above reactants are added together, mixed well, placed into acontainer of water at 75° C. and slow cooled to 42° C. Exposure to 300nm UV light for 10 minutes initiates a cross-linking event covalentlybinding the biotinylated oligos to the arms of the DNA dendrimer. Anadditional 1.1 uL of psoralen saturated solution in ethanol is added andthe reactants are exposed to an additional 2.5 minute exposure to 300 nmUV light. Non-cross-linked oligonucleotides are removed via the use of asize exclusion spin column or equivalent method.

For dendrimers containing more or less capture sequences, the quantityof “strand ligate” (reactant 2 above) can be varied accordingly.

A mouse anti-hCG targeting antibody (DCN, Carlsbad, Calif.) wasconjugated to the cplCap03 DNA oligonucleotide, which is complementaryto the Cap03 DNA oligonucleotide previously ligated to thedendrimer-bound “strand ligate”. The cplCap03 DNA oligonucleotide iscovalently conjugated to the anti-mouse IgG antibody using standardcross-linking condensation conjugation chemistry. This antibody-oligoconjugate may be used as a separate reagent in the lateral flow assay,or may be pre-combined with the DNA dendrimer via the hybridization ofthe antibody-bound cplCap03 oligonucleotide to the complementary Cap03sequence on the peripheral “arms” of the dendrimer. This hybridizationsite comprises 31 base pairs and has a melting temperature of greaterthan 65° C., thereby providing a stable complex of dendrimer bound withantibody at physiological temperatures and conditions (FIG. 4).

Performance of the POC Lateral Flow Assay Utilizing DNA Dendrimers asSignal Amplifiers:

The following materials were required:

-   -   1. Lateral flow strips consisting of a) nitrocellulose membranes        (HF180, EMD Millipore, Billerica, Mass.) and b) wick material.        The nitrocellulose membranes were striped with an anti-hCG alpha        antibody at a concentration suitable for lateral flow        immunoassay applications for use as a test line as well as an        additional stripe of an anti-mouse IgG antibody for use as a        control line. All components were appropriately blocked and        prepared for standard lateral flow immunoassays.    -   2. Liquid (aqueous) samples containing known amounts of hCG.    -   3. Mouse anti-hCG (beta) antibody-oligo conjugate for use as a        targeting molecule for dendrimer binding, in solution.    -   4. DNA dendrimer, biotinylated and containing the Cap03 capture        sequence (as described above), in an aqueous buffer solution.    -   5. Streptavidin-conjugated colloidal gold nanoparticles (40 nm        nominal size), in solution (Diagnostics Consulting Network        (DCN), Carlsbad, Calif.).    -   6. Phosphate buffered saline (PBS) with 0.05% Tween-20 (T) and        0.5% BSA (B) (PBS-TB).    -   7. Mouse anti-hCG colloidal gold conjugate (40 nm nominal size),        in solution (DCN).    -   8. 96 well polystyrene microtiter plates with flat bottom wells.

Assay procedure (multi-step):

-   -   1. All reagents were diluted using PBS-TB.    -   2. Into the first row of a 96 well microtiter plate, 15 μl of        the appropriate amount of hCG antigen (range starting at 100 mIU        with two-fold serial dilutions down to 0.024 mIU) were        dispensed.    -   3. Into the second row, 15 μl of the mouse anti-hCG        antibody-oligonucleotide conjugate at the correct dilution        (1:100, determined empirically) were dispensed.    -   4. Into the third row, 15 μl of dendrimer diluted to 13.3 ng/μl        (for a total final working amount of dendrimer per well/per        lateral flow strip to be 200 ng) were dispensed.    -   5. Into the fourth row, 20 μl of streptavidin-colloidal gold        conjugate diluted to 1.0 OD were dispensed.    -   6. The strips were placed into the wells of row 1 (containing        the hCG antigen) and the strips were leaned towards the rear of        the plate so that the bottom of the strip remained in contact        with the solution at the front of the well.    -   7. Once all the liquid in the wells of the first row had been        wicked into all of the strips, the strips were moved to the next        row of wells. This process was repeated for each strip into        succeeding wells until all liquid in each well has been absorbed        by the corresponding strip.    -   8. After all strips had been moved successfully through all the        wells, the visual score of the each test line was recorded (as        per a standard lateral flow visual scoring guide as per DCN        (FIG. 13)). A visible reddish or brown-reddish line at both the        test and control lines indicated a valid positive result (the        successful detection of hCG in the sample). A visible control        line only indicated a valid negative result (no detection of hCG        in the sample). The lack of any visible control line indicated        an invalid test and the results were discarded.    -   9. To set up a standard assay, follow the above procedure and        replace both the mouse anti-hCG antibody-oligo conjugate and        3DNA dendrimer with 15 μl of PBS-T and replace the streptavidin        colloidal gold conjugate with a mouse anti-hCG colloidal gold        conjugate (diluted to a working concentration of 1.0 OD).

Results of Utilizing a 3DNA Dendrimer in a Direct Sandwich POC LateralFlow hCG Assay (Wet Assay Format):

After performing the above described assay on hCG samples that rangefrom 100 mIU down to 0.024 mIU in two-fold serial dilutions, it wasobserved that a limit of detection (LOD) of 0.20-0.40 mIU hCG wasachieved for the dendrimer assay, compared to a LOD of 3.1-6.2 mIU forthe non-dendrimer standard hCG assay, resulting in a 8-32 foldimprovement of sensitivity when using the DNA dendrimer as a signalamplifier (FIG. 5).

Example 2 Use of a 3DNA Dendrimer and an Anti-Mouse IgG TargetingAntibody-Oligo Conjugate to Improve Sensitivity in an all Liquid(“Wet”), Indirect Sandwich POC Lateral Flow Assay

The following materials were required:

-   -   1. Lateral flow strips as described in Example 1.    -   2. Liquid (aqueous) samples containing hCG antigen as described        in Example 1.    -   3. Mouse anti-hCG (beta) antibody to be used as a primary        antibody (1:1000 dilution, determined empirically) (DCN).    -   4. Anti-mouse antibody-oligo conjugate for use as a targeting        molecule for dendrimer binding, in solution.    -   5. 3DNA biotinylated dendrimer, in solution.    -   6. Streptavidin-conjugated colloidal gold nanoparticles, as        described in Example 1.    -   7. PBS-TB, as described in Example 1.    -   8. Mouse anti-hCG colloidal gold conjugate, as described in        Example 1.    -   9. 96 well polystyrene microtiter plates as described in Example        1.

Assay procedure (multi-step): The assay was performed as in Example 1,except for the following steps:

-   -   3. Into the second row, dispense 15 μl of the mouse anti-hCG        antibody at the correct dilution (1:1000, determined        empirically).    -   4. Into the third row, dispense 15 μl of the anti-mouse        antibody-oligo conjugate at the correct dilution (1:18,        determined empirically).    -   5. Into the fourth row, dispense 15 μl of dendrimer diluted to        13.3 ng/μl (for a total final working amount of dendrimer per        well/per lateral flow strip to be 200 ng).    -   6. Into the fifth row, dispense 20 μl of streptavidin-colloidal        gold conjugate diluted to 1.0 OD.    -   7. The remaining steps were performed in a manner identical to        steps 7-9 of Example 1.

Results of utilizing a DNA dendrimer in an indirect sandwich POC lateralflow hCG assay (wet assay format): After performing the above describedassay on hCG samples that range from 100 mIU down to 0.024 mIU intwo-fold serial dilutions, it was observed that a limit of detection(LOD) of 0.10-0.20 mIU hCG was achieved for the dendrimer assay,compared to a LOD of 3.1-6.2 mIU for the non-dendrimer standard hCGassay, resulting in a 16-64 fold improvement of sensitivity when usingthe DNA dendrimer as a signal amplifier (FIG. 6).

Example 3 Effect of Various Physical Characteristics of DNA Dendrimerson Performance in Direct Sandwich POC Lateral Flow Assays as Performedin Example 1

Materials required: as in Example 1, except different DNA dendrimerswere utilized as described below.

Assay procedure (multi-step): the assay was performed as in Example 1except that different DNA dendrimers were used as described below.

Effect of Number of Capture Sequences

The antibody-oligo conjugate, capable of binding either directly orindirectly to analyte in a POC assay, “bridges” or binds to a DNAoligonucleotide “capture” sequence previously incorporated into a DNAdendrimer signal amplifier. The number of oligonucleotide “capture”sequences manufactured into the DNA dendrimer (via hybridization andcrosslinking of said arm) can be varied. DNA dendrimers containing ˜60,˜120 or ˜160 “capture” sequences were synthesized as describedpreviously. These DNA dendrimers were then utilized in a direct sandwichPOC lateral flow assay (“wet” format) as described in Example #1.

It was observed that as the number of capture sequences are increased, alower LOD was achieved in the hCG detection assay. As sensitivityincreased to the highest levels, non-specific binding (NSB) was observedin the assay, which was partially controlled via the use of variousreaction buffers, detergents and other reagents. These results supportedthat a higher number of antibody-oligo conjugate binding sites on theDNA dendrimers improved the sensitivity of the assay, likely viaimprovement of reaction kinetics via the increased likelihood of contactbetween the analyte, the antibody-oligo conjugate and the DNA dendrimersignal amplifier.

Effect of Number of Labels

The total number of label moieties (e.g., biotin) was varied on the DNAdendrimers. DNA dendrimers were synthesized with ˜1,440, ˜960, ˜480, and˜240 biotins. It was observed that DNA dendrimers containing ˜960biotins performed best, followed by ˜1,440, ˜480 and ˜240 biotinsrespectively. The poorer sensitivity of the DNA dendrimer with ˜1,440biotins was unexpected and may have been due to the somewhat larger sizeof this molecular reagent as compared to the other dendrimers, which mayhave kinetically disfavored the maximally labeled dendrimer in theimmunoassay.

Effect of Size

DNA dendrimers can be synthesized to be of various physical sizes, whichmay have an effect on the performance of these molecules in a POClateral flow system. Given that molecule size and structure contributesto the reaction kinetics of immunoassays, it is postulated that thelargest DNA dendrimers, while having the most label moieties, might bekinetically disfavored in the lateral flow assay, while smallerdendrimers containing fewer labels might perform as well or better inthe assay due to better reaction kinetics of the smaller molecules. Asmentioned above, the largest dendrimer with ˜1,440 biotin label moietiesperformed more poorly than the dendrimer with ˜960 biotins. The ˜1,440biotin dendrimer contains about 81,500 DNA nucleotides, compared toabout 69,900 DNA nucleotides for the ˜960 biotin dendrimer, a 16.6%difference in total mass. We believe this mass differential may beresponsible for the difference in performance between these molecules,notwithstanding the higher number of biotin moieties on the largerdendrimer. Further, additional DNA dendrimers of other sizes have beentested and were found to generate higher or lower signal amplificationresults that were not directly proportional to the difference in sizesbetween the varying dendrimers.

Example 4 Reduction of Non-Specific Binding (NSB) in a Direct SandwichPOC Lateral Flow Assay

Materials required: as in Example 1, except different PBS buffers wereutilized as described below.

Assay procedure (multi-step): the assay was performed as in Example 1except that different PBS buffers were used as described below.

In order to reduce or eliminate non-specific binding from POC lateralflow assays which utilize DNA dendrimers, a buffer matrix was devisedand tested. Nine phosphate buffered saline (PBS) buffers were tested,which consisted of varying percentages of bovine serum albumin (BSA,Sigma) or Tween-20 (Sigma).

The buffers were composed of:

#1: 1×PBS, 0.1% BSA, 0.01% Tween-20 #2: 1×PBS, 0.1% BSA, 0.1% Tween-20#3: 1×PBS, 0.1% BSA, 0.5% Tween-20 #4: 1×PBS, 0.25% BSA, 0.01% Tween-20#5: 1×PBS, 0.25% BSA, 0.1% Tween-20 #6: 1×PBS, 0.25% BSA, 0.5% Tween-20#7: 1×PBS, 0.5% BSA, 0.01% Tween-20 #8: 1×PBS, 0.5% BSA, 0.1% Tween-20#9: 1×PBS, 0.5% BSA, 0.5% Tween-20 Results of Varying Buffers on NSB:

Lateral flow assays run in Buffer #7 resulted in the best specificsignal and the lowest NSB. Unexpectedly, the presence of relatively highpercentages of Tween-20 (>0.01%) in the PBS buffers resulted in anincrease of NSB and those buffers were deselected for use in the lateralflow assays.

Example 5 Enhancement of Sensitivity (Specific Signal) in a DNADendrimer Direct Sandwich POC Lateral Flow Assay by Addition of SpecificChemical Agents

Prior studies have shown that various types of additive chemicals havean enhancing effect on the sensitivity (signal) produced when using DNAdendrimers in various assay systems (e.g., DNA/RNA microarrays). Thechemicals used are typically of volume-excluding polymers such aspolyethylene glycol (PEG) or other highly branched polysaccharidemolecules. Two different lateral flow PBS buffer formulations: a) 1XPBS, 0.5% Tween-20 and b) 1X PBS, 0.5% BSA, and 0.01% Tween-20 (Buffer#7 from Example #4) were used for these experiments.

Materials required: as in Example 1, except the following chemicals wereadded to the DNA dendrimer dilution buffer at various concentrations:

PEG 3350 PEG 6000 PEG 8000 Ficoll (MW70,000)

Dextran sulfate

Sucrose

Assay procedure (multi-step): the assay was performed as in Example 1.

Results of Testing Enhancement Chemicals:

All chemicals were tested at various concentrations to determine ifsensitivity was improved in an hCG titration assay (see Example 1),while also measuring NSB. Of the chemicals tested, dextran sulfate at aconcentration of 0.75% (vol:vol) provided significant improvement ofsensitivity, averaging 2-4 fold more sensitivity than the dextransulfate free dendrimer assay, with no appreciable increase of NSB. Theother chemical reagents provided little or no improvement of sensitivityor demonstrated unacceptable levels of NSB, or both, contrary to resultsachieved in other assay formats (e.g., ELISA).

Example 6 Use of Different Types of Colloidal Gold Particles in a DNADendrimer POC Lateral Flow Assay

Multiple sized streptavidin or neutravidin conjugated gold particleswere tested in a direct sandwich POC lateral flow system with DNAdendrimers to ascertain which particles provided the most sensitivitywith little or no NSB.

Materials required: as in Example 1, except the following gold particleswere tested at an 1.0 OD final concentration:

Streptavidin-gold conjugate, 20 nmStreptavidin-gold conjugate, 30 nmStreptavidin-gold conjugate, 40 nmStreptavidin-gold conjugate, 80 nmNeutravidin-gold conjugate, 40 nmAnti-biotin antibody-gold conjugate, 40 nmGold particles conjugated with DNA oligonucleotides complementary to DNAdendrimer sequences, 20 nm

Results of Testing Various Gold Particles:

The best sensitivity, signal, and low NSB were achieved with both the 30nm and 40 nm streptavidin-gold particles. Unexpectedly, the 80 nmstreptavidin-gold particles produced a relatively weak signal, perhapsindicating a poorer ability of the larger particles to migrateeffectively through the lateral flow membrane. The gold particlesderivatized with the DNA oligonucleotides complementary to the DNAdendrimer worked well, achieving sensitivity equal to or exceeding the20 nm streptavidin-gold particle (a larger oligonucleotide conjugatedparticle was not available, although we believe a larger particle wouldperform even better than the tested 20 nm particle).

Example 7 Use of a DNA Dendrimer and Other Assay Components in a Partialor Fully Dried-Down, Direct or Indirect Sandwich POC Lateral Flow hCGAssay

Materials required: as in Example 1 and Example 2, except for thefollowing:

-   -   1. 3× Dry Down Solution (15% Trehalose (Fisher), 60% Sucrose        (Sigma) solution, formulated in DDH₂O).    -   2. 37° C. forced air oven.    -   3. Glass test tubes (12×75 mm).

Drying-Down Reagent Preparation:

Anti-Mouse Antibody-Oligo Conjugate

The anti-mouse antibody-oligo conjugate was prepared at 1:18 dilutionwith a final concentration of 5% Trehalose and 20% Sucrose (startingwith 3× Dry Down Solution diluted with 1×PBS with 0.5% BSA and 0.01%Tween-20).

DNA Dendrimer

A total mass of 65-200 ng of DNA dendrimer (per 5 uL per strip) wasdiluted in a final solution containing 5% Trehalose and 20% Sucrose(starting with 3× Dry Down Solution diluted with 1×PBS with 0.5% BSA and0.01% Tween-20).

Streptavidin Colloidal Gold

Streptavidin-gold (SA-gold) conjugate was diluted to a concentration of1.5 OD in 5% Trehalose and 20% Sucrose (starting with 3× Dry DownSolution diluted with 1×PBS with 0.5% BSA and 0.01% Tween-20).

Drying-Down Procedure:

Drying-Down a Single Reagent (Antibody-Oligo Conjugate, DNA Dendrimer,or SA-Gold):

-   -   1. Pipette 50 of properly prepared reagent onto the middle of        the Porex conjugate pad on the lateral flow strip.    -   2. Place strips in 37° C. forced air oven for 30 minutes.

Drying-Down Two Reagents (Antibody-Oligo Conjugate or DNA Dendrimer):

-   -   1. Pipette 50 of the first reagent onto the Porex conjugate pad        approximately 1 cm from the bottom of the pad on the lateral        flow strip.    -   2. Pipette 50 of the second reagent onto the bottom of the Porex        conjugate pad.    -   3. Place strips in 37° C. forced air oven for 30 minutes.

Drying-Down Three Reagents (Antibody-Oligonucleotide Conjugate, DNADendrimer, and SA-Gold):

-   -   1. Pipette 50 of the first reagent onto the Porex pad        approximately 1 cm from the bottom of the pad.    -   2. Pipette 50 of the second reagent onto the middle of the Porex        pad.    -   3. Pipette 50 of the third reagent onto the bottom of the Porex        pad.    -   4. Place strips in 37° C. forced air oven for 30 minutes.

Assay Procedures:

A direct sandwich assay containing only one dried-down reagent:

-   -   1. Lateral flow strips were placed into glass tubes containing        65 μl of the appropriate amount of sample containing hCG        antigen.    -   2. After the sample liquid was completely absorbed into the        strip, the strips were moved to glass tubes containing 15 μl of        either antibody-oligo conjugate or DNA dendrimer.    -   3. After all liquid was completely absorbed into the strips, the        strips were moved into glass tubes containing 20 μl of SA-gold        (providing the gold is not dried down on the strip).    -   4. After SA-gold conjugate was completely absorbed into the        lateral flow strip, the strips were moved to glass tubes        containing 35 μl of 1×PBS-TB buffer.    -   5. After completion of the assay, scores were recorded for as        per the visual scoring guide.    -   6. The standard comparative assay substituted anti-hCG colloidal        gold for SA-gold conjugate and did not require the use of the        DNA dendrimer and antibody-oligo conjugate reagents.

A direct sandwich assay containing two dried-down reagents: Sameprocedure as immediately above, except that one of the “wet” reagentsteps was eliminated, resulting in a two step assay.

A direct sandwich assay containing three dried-down reagents: Sameprocedure as immediately above, except that one of the “wet” reagentsteps was eliminated, resulting in a one step assay.

Results of Utilizing Dried Reagents (Including DNA Dendrimers) in aDirect Sandwich POC Lateral Flow hCG Assay

After performing the above described assay on hCG samples that rangefrom 100 mIU down to 0.024 mIU in two-fold serial dilutions, it wasobserved that a limit of detection (LOD) of 0.01-0.40 mIU hCG wasachieved for the dendrimer assay, compared to a LOD of 3.1-6.2 mIU forthe non-dendrimer standard hCG assay, resulting in a 8-64 foldimprovement of sensitivity when using the DNA dendrimer as a signalamplifier (FIG. 7).

Indirect sandwich lateral flow assays containing up to four dried downreagents may also be performed in a manner similar to the above, withsimilar results.

Example 8 Use of a Fluorescent DNA Dendrimer and Other Assay Componentsin a Direct Sandwich POC Lateral Flow hCG Assay

DNA dendrimers directly labeled with fluorescent dyes were used in POClateral flow hCG assay (“wet”). The DNA dendrimer was directly labeledwith ˜960 fluorescent Oyster 650 dyes and was substituted for thebiotinylated DNA dendrimer and SA-gold conjugate used in Example 1.Otherwise the assay was performed as described in Example 1. Resultsindicated that sensitivity was equal to or better than the sensitivityachieved using the SA-gold visible signal assay format (FIG. 8).

Example 9 Use of a DNA Dendrimer and an Anti-hCG Mouse MonoclonalTargeting Antibody-Oligo Conjugate to Improve Sensitivity in an allLiquid (“Wet”), Direct Sandwich POC Lateral Flow Assay, where the DNADendrimer Contains Label Moieties Non-Covalently Bound

This example is identical to Example 1, except for the followingdendrimer manufacturing process describing the binding of labeledoligonucleotides and strand ligate to the dendrimer structure:

Add to a microfuge tube the following components:

4 layer DNA dendrimer 1000 ng (1 ug) strand ligate (from above) 500 ngas strand (total synthesis 10 uL) c(+) 2x biotin oligo (35mer) 452.4 ng(complementary to dendrimer “c arm” N3(−) #1 2x biotin oligo (18mer)291.9 ng (complementary to “strand 3 ligate”) N3(−) #2 2x biotin oligo(19mer) 243.2 ng (complementary to “strand 3 ligate”) N3(−) #3 2x biotinoligo (26mer) 316.2 ng (complementary to “strand 3 ligate”) 5M NaCl 1.1uL (0.2M Final) Nuclease free Water to 28 uL total volume

The above reactants are added together, mixed well, placed into acontainer of water at 75° C. and slow cooled to room temperature toallow for the hybridization of the strand ligate and labeled oligos tothe dendrimer structure. Non-hybridized oligonucleotides are removed viathe use of a size exclusion spin column or equivalent method.

Results for this example were consistent with the results reported inExample 1.

Example 10 A Study of DNA Dendrimer Release from a Lateral Flow Device

Experimental Design: Materials

The materials used in this study are listed below:

-   -   Radiolabeled (³²P) DNA dendrimer containing biotin    -   Anti-hCG β antibody-oligo conjugate    -   Dry-down Buffer (containing sucrose and trehalose) (3× Dry Down        Solution in Example 7 [00182])    -   Conjugate pads (Porex, Porex brand)    -   Lateral flow strips containing either HF90 or HF180        nitrocellulose (Millipore brand)    -   hCG Antigen    -   Running Buffer (Buffer No. 7 listed in Example 4 [00173])    -   Streptavidin gold conjugate (40 nm particle)

Experimental Design: Methods

The radiolabeled DNA dendrimer (without antibody-oligo conjugate) wasmixed with the dry-down buffer onto the hCG assay lateral flow conjugatepads and affixed to both HF90 and HF180 nitrocellulose membranes. Theradiolabeled DNA dendrimer (combined with antibody-oligo conjugate) wasmixed with the dry-down buffer onto the hCG assay lateral flow conjugatepads and affixed to HF90 nitrocellulose membranes. The following aqueoussolutions were applied to the proximal end of the conjugate pad(containing the dried down dendrimer) of the hCG lateral flow strips toassess the efficiency of release of the dendrimer from the conjugate padand to identify where the dendrimer migrated to on the lateral flowdevice:

No buffer control;

65 uL of Running Buffer with diluted hCG antigen;

65 uL of Running Buffer with diluted hCG antigen followed by 20 uL ofgold particles;

65 uL of Running Buffer with diluted hCG antigen followed by 20 uL ofgold particles followed by 35 uL of Running Buffer (wash step).

The lateral flow devices were disassembled and each region (as indicatedFIG. 9) was evaluated for the quantity of ³²P by counting CPM in ascintillation counter (Beckman LS6500).

The results of dendrimer dried without antibody-oligo conjugate and runon HF90 nitrocellulose containing hCG lateral flow assays are shown inTable 1 and FIG. 10. As shown in Table 1 and FIG. 10, about 73% ofdendrimer was released and migrated to the wick with a total aqueousvolume of 65 uL; about 91% of dendrimer was released and migrated to thewick with a total aqueous volume of 85 uL; and about 92% of dendrimerwas released and migrated to the wick with a total aqueous volume of 120uL.

TABLE 1 Strip Total CPM % of CPM in HF90 NC Region CPM in Strip StripRegions Strip 1: Region 1 163451.40 163631.44 99.89 % in Region 1 No LF,Region 2 49.01 0.03 % in Region 2 only Region 3 46.01 0.03 % in Region 3spotted Region 4 40.01 0.02 % in Region 4 material Region 5 45.01 0.03 %in Region 5 Strip 2: Region 1 18385.45 131967.94 13.93 % in Region 1Only Region 2 5401.75 4.09 % in Region 2 Antigen + Region 3 5181.77 3.93% in Region 3 Buffer (65 Region 4 6682.42 5.06 % in Region 4 uL total)Region 5 96316.55 72.98 % in Region 5 Strip 3: Region 1 4421.79169521.55 2.61 % in Region 1 Antigen + Region 2 2015.86 1.19 % in Region2 Buffer (65 Region 3 3254.81 1.92 % in Region 3 uL) + Region 4 5412.593.19 % in Region 4 SA Gold Region 5 154416.50 91.09 % in Region 5 (20uL) Strip 4: Region 1 2366.23 151466.62 1.56 % in Region 1 Antigen +Region 2 1266.41 0.84 % in Region 2 Buffer (65 Region 3 2874.99 1.90 %in Region 3 uL) + Region 4 5673.39 3.75 % in Region 4 SA Gold Region 5139285.60 91.96 % in Region 5 (20 uL) + Chase (35 uL)

The results of dendrimer dried without antibody-oligo conjugate and runon HF180 nitrocellulose containing hCG lateral flow assays are shown inTable 2 and FIG. 11. As shown in Table 2 and FIG. 11, about 61% ofdendrimer was released and migrated to the wick with a total aqueousvolume of 65 uL; about 81% of dendrimer was released and migrated to thewick with a total aqueous volume of 85 uL; and 83% of dendrimer wasreleased and migrated to the wick with a total aqueous volume of 120 uL.

TABLE 2 HF180 Strip Total CPM % of CPM in NC Region CPM in Strip StripRegions Strip 1: Region 1 157061.50 157273.50 99.87 % in Region 1 No LF,Region 2 48.00 0.03 % in Region 2 only Region 3 74.00 0.05 % in Region 3spotted Region 4 47.00 0.03 % in Region 4 material Region 5 43.00 0.03 %in Region 5 Strip 2: Region 1 33667.92 157245.41 21.41 % in Region 1Only Region 2 9742.48 6.20 % in Region 2 Antigen + Region 3 8202.24 5.22% in Region 3 Buffer (65 Region 4 9762.86 6.21 % in Region 4 uL total)Region 5 95869.91 60.97 % in Region 5 Strip 3: Region 1 7532.53154336.96 4.88 % in Region 1 Antigen + Region 2 3954.75 2.56 % in Region2 Buffer Region 3 7933.05 5.14 % in Region 3 (65 uL) + Region 4 10284.136.66 % in Region 4 SA Gold Region 5 124632.50 80.75 % in Region 5 (20uL) Strip 4: Region 1 2170.97 140307.01 1.55 % in Region 1 Antigen +Region 2 1323.48 0.94 % in Region 2 Buffer Region 3 11185.54 7.97 % inRegion 3 (65 uL) + Region 4 8764.52 6.25 % in Region 4 SA Gold Region 5116862.50 83.29 % in Region 5 (20 uL) + Chase (35 uL)

The results of dendrimer dried with antibody-oligo conjugate andperformed on HF90 nitrocellulose containing hCG lateral flow assays areshown in Table 3 and FIG. 12. As shown in Table 3 and FIG. 12, about 87%of dendrimer was released and migrated to the wick with a total aqueousvolume of 65 uL; about 76% of dendrimer was released and migrated to thewick with a total aqueous volume of 85 uL; and about 79% of dendrimerwas released and migrated to the wick with a total aqueous volume of 120uL.

TABLE 3 HF90 NC + Precombined Total Den and Strip CPM % of CPM in ConjRegion CPM in Strip Strip Regions Strip 1: Region 1 130796.40 131020.5679.93 % in Region 1 No LF, only Region 2 39.02 0.02 % in Region 2spotted Region 3 43.03 0.03 % in Region 3 material Region 4 93.07 0.06 %in Region 4 Region 5 49.04 0.03 % in Region 5 Strip 2: Region 1 9948.11136194.88 7.54 % in Region 1 Only Region 2 4483.76 3.40 % in Region 2Antigen + Region 3 2855.79 2.16 % in Region 3 Buffer (65 Region 44544.02 3.44 % in Region 4 uL total) Region 5 114363.20 86.66 % inRegion 5 Strip 3: Region 1 1805.68 135164.23 1.07 % in Region 1Antigen + Region 2 1264.07 0.75 % in Region 2 Buffer Region 3 1404.720.83 % in Region 3 (65 uL) + Region 4 2037.06 1.20 % in Region 4 SA GoldRegion 5 128652.70 75.89 % in Region 5 (20 uL) Strip 4: Region 1 1521.61124861.45 1.00 % in Region 1 Antigen + Region 2 544.60 0.36 % in Region2 Buffer Region 3 1076.22 0.71 % in Region 3 (65 uL) + Region 4 2077.421.37 % in Region 4 SA Gold Region 5 119641.60 78.99 % in Region 5 (20uL) + Chase (35 uL)

The above results show that 65 uL of aqueous solution released 73%(HF90) to 61% (HF180) of the dried dendrimer with migration through thelength of the nitrocellulose membrane with final deposition in theabsorbant wick, 85 uL of aqueous solution released 91% (HF90) to 81%(HF180) of the dried dendrimer with migration through the length of thenitrocellulose membrane with final deposition in the absorbant wick, and120 uL of aqueous solution released 92% (HF90) to 83% (HF180) of thedried dendrimer with migration through the length of the nitrocellulosemembrane with final deposition in the absorbant wick.

The HF90 nitrocellulose membrane appeared to effect slightly moreefficient release of dried dendrimer as compared to the HF180 membrane.Dendrimer precombined with antibody-oligo conjugate tended to releaseslightly less efficiently from the conjugate pad as compared todendrimer reagent dried alone on the conjugate pad. In summary, DNADendrimer dried on the conjugate pad (Porex) is easily and efficientlyreleased with the application of minimal volumes of aqueous solutionsduring a typical lateral flow assay.

Example 11 A Study of the Effect of the Number of Dendrimer CaptureSequences on Dendrimer Augmented Lateral Flow Assays

Experimental Design: Materials

The materials used in this study are listed below:

-   -   4 layer DNA dendrimers containing ˜900 biotin and various        numbers of capture sequences:        -   “1×”=approximately 18 capture sequences per dendrimer        -   “2×”=approximately 35 capture sequences per dendrimer        -   “4×”=approximately 71 capture sequences per dendrimer        -   “6×”=approximately 95 capture sequences per dendrimer        -   “8×”=approximately 115 capture sequences per dendrimer    -   Anti-hCG β antibody-oligo conjugate    -   Dry-down buffer (containing sucrose and trehalose) (3X Dry Down        Solution in Example 7 [00182])    -   Conjugate pads (Porex, Porex brand)    -   Lateral flow strips containing HF180 nitrocellulose (Millipore        brand) and anti-hCG alpha antibody striped down at a        concentration of 1 mg/mL    -   hCG Antigen    -   Running buffer (Buffer No. 7 listed in Example 4 [00173])    -   Streptavidin gold conjugate (40 nm particle)

Experimental Design: Methods

DNA dendrimers with varying numbers of capture sequences were run in astandardized hCG assay lateral flow assay as follows:

-   -   1. DNA dendrimer, anti-hCG beta antibody-oligo conjugate and        streptavidin-gold conjugate were dried down on the conjugate pad        of the hCG lateral flow assay.    -   2. Each lateral flow strip was placed conjugate pad first into        100 uL of Buffer No. 7 listed in Example 4 ([00173]) containing        15, 3 and 0.6 mIU of hCG antigen. The aqueous running buffer was        allowed to run up the lateral flow strip for 20 minutes, and        then the reaction at the test line containing an anti-hCG alpha        antibody was visually read as per the DCN scoring system (FIG.        13).

The results of this study are shown in FIG. 14. As shown in FIG. 14,there appears to be a direct correlation of the number of capturesequences on a 900 biotin 4 layer dendrimer and the level of signalamplification in an hCG lateral flow assay, with 6× and 8× (95 and 115capture sequences per dendrimer respectively) providing the highestlevel of signal amplification.

The above examples are included for illustrative purposes only and arenot intended to limit the scope of the invention. Many variations tothose described above are possible. Since modifications and variationsto the examples described above will be apparent to those of skill inthis art, it is intended that this invention be limited only by thescope of the appended claims.

1. A test device for detecting an analyte in a liquid sample, whichdevice comprises a porous matrix that comprises a test zone on saidporous matrix, said test zone comprising a test reagent that binds to ananalyte or another binding reagent that binds to said analyte, or is ananalyte or an analyte analog that competes with an analyte in saidsample for binding to a binding reagent for said analyte, wherein aliquid sample flows laterally along said test device and passes saidtest zone to form a detectable signal to indicate presence, absenceand/or amount of said analyte in said liquid sample, the formation ofsaid detectable signal requires the use of a detectable label and a DNAdendrimer, said DNA dendrimer comprises a first component that linkssaid DNA dendrimer to a binding reagent linkable to said DNA dendrimer,an analyte linkable to said DNA dendrimer, or an analyte analog linkableto said DNA dendrimer, and a second component that links said DNAdendrimer to said detectable label linkable to said DNA dendrimer, andwherein: a) said analyte is not a polynucleotide; or b) said DNAdendrimer comprises from about 1 to about 324, preferably from about 60to about 324, from about 80 to about 300 or from about 100 to about 200,said first components; or c) said DNA dendrimer, before said test deviceis used, is dried on a location on said test device upstream from saidtest zone. 2-7. (canceled)
 8. A method for detecting an analyte in aliquid sample, which method comprises: a) contacting a liquid samplewith the test device of claim 1, wherein the liquid sample is applied toa site of the test device upstream of the test zone; b) transporting ananalyte, if present in the liquid sample, a detectable label and a DNAdendrimer to the test zone; and c) assessing the presence, absence,and/or amount of a signal generated by the detectable label at the testzone to determining the presence, absence and/or amount of the analytein the liquid sample. 9-12. (canceled)
 13. A test device for detectingan analyte in a liquid sample, which device comprises a porous matrixthat comprises a test zone on said porous matrix, said test zonecomprising a test reagent that binds to an analyte or another bindingreagent that binds to said analyte, or is an analyte or an analyteanalog that competes with an analyte in said sample for binding to abinding reagent for said analyte, wherein a liquid sample flowslaterally along said test device and passes said test zone to form adetectable signal to indicate presence, absence and/or amount of saidanalyte in said liquid sample, the formation of said detectable signalrequires the use of a DNA dendrimer linked to a detectable labelnon-covalently, and said DNA dendrimer comprises a component that linkssaid DNA dendrimer to a binding reagent linkable to said DNA dendrimer,an analyte linkable to said DNA dendrimer, or an analyte analog linkableto said DNA dendrimer, and wherein: a) said analyte is not apolynucleotide; or b) said DNA dendrimer comprises from about 1 to about324, preferably from about 60 to about 324, from about 80 to about 300or from about 100 to about 200, said first components; or c) said DNAdendrimer, before said test device is used, is dried on a location onsaid test device upstream from said test zone. 14-21. (canceled)
 22. Amethod for detecting an analyte in a liquid sample, which methodcomprises: a) contacting a liquid sample with the test device of claim13, wherein the liquid sample is applied to a site of the test deviceupstream of the test zone; b) transporting an analyte, if present in theliquid sample, a detectable label and a DNA dendrimer to the test zone;and c) assessing the presence, absence, and/or amount of a signalgenerated by the detectable label at the test zone to determining thepresence, absence and/or amount of the analyte in the liquid sample.23-25. (canceled)
 26. The test device of claim 1, wherein the DNAdendrimer, before the test device is used, is dried on a location on thetest device upstream from the test zone, and at least about 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% ofthe dried DNA dendrimer is capable of being moved to the test zone by aliquid. 27-32. (canceled)
 33. The test device of claim 26, wherein theliquid has a volume of at least about 20 ul, 25 ul, 30 ul, 35 ul, 40 ul,45 ul, 50 ul, 55 ul, 60 ul, 65 ul, 70 ul, 75 ul, 80 ul, 85 ul, 90 ul, 95ul, 100 ul, 110 ul, 120 ul, 150 ul, 200 ul, 300 ul, 400 ul, 500 ul, or avolume larger than 500 ul.
 34. The test device of claim 1, wherein theDNA dendrimer comprises: a) an one-layer DNA dendrimer that comprisesfrom about 1-2, 2-4 or 4-8 of the first and/or second component(s); b) atwo-layer DNA dendrimer that comprises from about 1-4, 4-8 or 8-18 ofthe first and/or second component(s); c) a three-layer DNA dendrimerthat comprises from about 2-6, 6-10, 10-18, 18-28, 28-38, 38-50 or morethan 50 of the first and/or second components; and/or d) a four-layerDNA dendrimer that comprises from about 15-25, 25-45, 45-60, 60-80,85-105, 105-130 or more than 130 of the first and/or second components.35. The test device of claim 34, wherein at least one of the firstcomponent(s) and the second component(s) is a polynucleotide and theother is a non-polynucleotide moiety.
 36. The test device of claim 34,wherein the DNA dendrimer comprises: a) an one-layer DNA dendrimer thatcomprises from about 1-2, 2-4 or 4-8 of the first component(s); b) atwo-layer DNA dendrimer that comprises from about 1-4, 4-8 or 8-18 ofthe first component(s); c) a three-layer DNA dendrimer that comprisesfrom about 2-6, 6-10, 10-18, 18-28, 28-38, 38-50 or more than 50 of thefirst components; and/or d) a four-layer DNA dendrimer that comprisesfrom about 15-25, 25-45, 45-60, 60-80, 85-105, 105-130 or more than 130of the first components.
 37. The test device of claim 36, wherein thefirst component(s) comprise polynucleotide(s).
 38. The test device ofclaim 37, wherein the polynucleotide(s) are DNA strand(s).
 39. A methodfor detecting an analyte in a liquid sample, which method comprises: a)contacting a liquid sample with the test device of claim 26, wherein theliquid sample is applied to a site of the test device upstream of thetest zone; b) transporting an analyte, if present in the liquid sample,a detectable label and a DNA dendrimer to the test zone; and c)assessing the presence, absence, and/or amount of a signal generated bythe detectable label at the test zone to determining the presence,absence and/or amount of the analyte in the liquid sample.
 40. Themethod of claim 39, wherein the signal generated by the detectable labelat the test zone is enhanced by: a) at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, 150%, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 folds or higher relative to a signalgenerated by the detectable label at the test zone using a DNA dendrimercomprising a smaller number of the first and/or second component(s); orb) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30,40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200 folds or higher relative to a signal generated by thedetectable label at the test zone without using a DNA dendrimer.
 41. Atest device for detecting an analyte in a liquid sample, which devicecomprises a porous matrix that comprises a test zone on said porousmatrix, said test zone comprising a test reagent that binds to ananalyte or another binding reagent that binds to said analyte, or is ananalyte or an analyte analog that competes with an analyte in saidsample for binding to a binding reagent for said analyte, wherein aliquid sample is capable of flowing laterally along said test device andpassing said test zone to form a detectable signal to indicate presence,absence and/or amount of said analyte in said liquid sample, wherein: a)the formation of said detectable signal requires the use of a detectablelabel and a DNA dendrimer, said DNA dendrimer comprises a firstcomponent that links said DNA dendrimer to a binding reagent linkable tosaid DNA dendrimer, an analyte linkable to said DNA dendrimer, or ananalyte analog linkable to said DNA dendrimer, and a second componentthat links said DNA dendrimer to said detectable label linkable to saidDNA dendrimer, or b) the formation of said detectable signal requiresthe use of a DNA dendrimer linked to a detectable label covalently ornon-covalently, and said DNA dendrimer comprises a third component thatlinks said DNA dendrimer to a binding reagent linkable to said DNAdendrimer, an analyte linkable to said DNA dendrimer, or an analyteanalog linkable to said DNA dendrimer, and wherein: c) said analyte isnot a polynucleotide; and/or d) said DNA dendrimer comprises from about1 to about 324, preferably from about 60 to about 324, from about 80 toabout 300 or from about 100 to about 200, said first components; and/ore) said DNA dendrimer, before said test device is used, is dried on alocation on said test device upstream from said test zone.
 42. A methodfor detecting an analyte in a liquid sample, which method comprises: a)contacting a liquid sample with the test device of claim 41, wherein theliquid sample is applied to a site of the test device upstream of thetest zone; b) transporting an analyte, if present in the liquid sample,a detectable label and a DNA dendrimer to the test zone; and c)assessing the presence, absence, and/or amount of a signal generated bythe detectable label at the test zone to determining the presence,absence and/or amount of the analyte in the liquid sample.
 43. A kit fordetecting an analyte in a liquid sample, which kit comprises: a) aporous matrix that comprises a test zone on said porous matrix, saidtest zone comprising a test reagent that binds to an analyte or anotherbinding reagent that binds to said analyte, or is an analyte or ananalyte analog that competes with an analyte in said sample for bindingto a binding reagent for said analyte; and b) a DNA dendrimer,wherein: 1) said DNA dendrimer comprises a first component that linkssaid DNA dendrimer to a binding reagent linkable to said DNA dendrimer,an analyte linkable to said DNA dendrimer, or an analyte analog linkableto said DNA dendrimer, and a second component that links said DNAdendrimer to said detectable label linkable to said DNA dendrimer, or 2)said DNA dendrimer is linked to a detectable label covalently ornon-covalently and comprises a third component that links said DNAdendrimer to a binding reagent linkable to said DNA dendrimer, ananalyte linkable to said DNA dendrimer, or an analyte analog linkable tosaid DNA dendrimer.
 44. The kit of claim 43, wherein the DNA dendrimercomprises from about 1 to about 324, preferably from about 60 to about324, from about 80 to about 300 or from about 100 to about 200, of thefirst or third components. 45-46. (canceled)
 47. The kit of any claim43, wherein at least two of: 1) the DNA dendrimer; 2) the first bindingreagent that binds to the analyte, the second binding reagent that bindsto another binding reagent that binds to the analyte, the analyte or theanalyte analog, each of the first binding reagent, second bindingreagent, analyte or analyte analog being linkable to the DNA dendrimer;and 3) the detectable label linkable to the DNA dendrimer; arecovalently bound or crosslinked to each other.
 48. The kit of any ofclaim 43, wherein the DNA dendrimer comprises from about 10 to about1,500, preferably from about 40 to about 1,500 or from about 900 toabout 1,100, the detectable label.
 49. The kit of any of claim 43,wherein the DNA dendrimer comprises from about 400 to about 80,000,preferably from about 4,000 to about 80,000, or from about 60,000 toabout 80,000, DNA nucleotides.
 50. The kit of any of claim 43, whereinthe DNA dendrimer comprises a one-layer, a two-layer, a three-layer or afour-layer structure, or a DNA dendrimer structure comprising at leastone monomeric unit as described in U.S. Pat. No. 6,274,723B1.
 51. Thekit of any of claim 43, which further comprises one or more of anantibody-oligo conjugate, an analyte capture antibody,streptavidin-label, e.g., gold particle, conjugate or other label,buffers, reagents, instructions etc.